inhibition thrombusformation by activated recombinant...

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Inhibition of Thrombus Formation byActivated Recombinant Protein C in a

Primate Model of Arterial ThrombosisAndras Gruber, MD, Stephen R. Hanson, PhD, Andrew B. Kelly, DVM, Betty S. Yan, PhD,

Nils Bang, MD, John H. Griffin, PhD, and Laurence A. Harker, MD

Activated protein C (APC) is an antithrombotic enzyme. The therapeutic potential of infusedhuman recombinant APC (rAPC) was studied in a primate model of platelet-dependentthrombosis. Eight baboons with chronic femoral arteriovenous shunts received rAPC infusionsfor 1 hour. The shunts were extended with 5-cm long, 4-mm-i.d. thrombogenic Dacron graftsegments for the time of infusion. The plasma level of the enzyme, the blood flow in the shunt,and the deposition of indium-ill-labeled platelets and iodine-125 fibrinogen on the graft weremeasured. The influence of rAPC infused at doses of 0.25 and 1.0 mg/kg-hr was compared withthe effects of control infusions of saline. Five of eight control grafts occluded within 60 minutes,whereas there was no change in the blood flow during rAPC infusion. Deposition of plateletswas inhibited by 13±10% and by 42±13% (mean±SEM) after 30 minutes of infusion at the twodoses, which gave rise to circulating rAPC plasma concentrations of 0.4 and 1.9 mg/l,respectively. Both doses significantly inhibited fibrin deposition in the graft. Circulatingplasma markers of thrombus formation and of fibrinolysis did not increase significantly duringrAPC infusion; measurements of bleeding time were also within normal limits. Thus, rAPC, likehuman plasma-derived APC, inhibited thrombus formation without impairing primaryhemostasis. (Circulation 1990;82:578-585)

Activated protein C (APC) is an antithromboticserine protease. It is generated from vitaminK-dependent plasma protein C (PC) by the

catalytic complex of thrombin and thrombomodulin.'PC circulates in blood at a plasma level of 4 mg/1.2APC acts by inhibiting thrombin formation by meansof enzymatic cleavage and destruction of coagulationfactors Va and VIIa, thus providing negative feed-back regulation of coagulation.3 The physiologicalimportance of APC is shown by clinical observationsthat 1) the incidence of heterozygous PC deficiency is

higher in patients with thrombophilia (4%)4.5 thanamong healthy individuals (0.4%)6; 2) there is a pos-

From the Committee on Vascular Biology and the Department ofMolecular and Experimental Medicine, Research Institute of ScrippsClinic (A.G., S.R.H., A.B.K, J.H.G., L.A.H.), La Jolla, Calif., andLilly Research Laboratories (B.S.Y., N.B.), Indianapolis, Ind.

Presented in part at the 61th Annual Scientific Sessions of theAmerican Heart Association, November 14-17, 1988, Washington,D.C.

Supported by National Institutes of Health grant HL-31950 andby the California Affiliate of the American Heart Association.Address for correspondence: Dr. John H. Griffin, Department

of Molecular and Experimental Medicine, BCR5, Research Insti-tute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla,CA 92037.

Received October 18, 1989; revision accepted April 10, 1990.

itive correlation between thrombotic events and het-erozygous PC deficiency in some thrombophilicfamilies7; 3) severe thrombophilia of homozygous pro-tein C-deficient infants is successfully controlled witheither repeated replacement of protein C or livertransplantation8,9; and 4) isolated acquired PC defi-ciency may be associated with serious thrombotic

See p 655

complications.10°11 Experimental results obtained invarious animal species indicate that purified plasma-derived APC is a potent and safe antithrombotic agentwhen used in pharmacological doses. The enzymeinhibits thrombus formation in microcirculatory,1213venous,14 and arterial15 thrombosis models.

In this study, we evaluated the effects of activatedrecombinant protein C (rAPC) in a nonhuman pri-mate model of arterial thrombosis and found thatinfusion of rAPC inhibited platelet-dependent throm-bus formation at plasma levels that did not impairprimary hemostasis.

MethodsExperimental protocol

Eight young, healthy male baboons (Papio anubis),weighing 9-13 kg and bearing chronic femoral arterio-

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Gruber et al Activated Recombinant Protein C for Thrombosis 579

venous shunts, were used in the experiments. Allanimal procedures, including preparation of theshunts, insertion of thrombogenic grafts, and detec-tion of thrombus formation, were performed asdescribed elsewhere.16,17 This model has been shownto provide reproducible results in repeated experi-ments in the same animal, that is, grafts may beevaluated at daily intervals for up to 4 days withequivalent results.16 All studies were approved by theinstitutional animal care and use committee inaccordance with federal guidelines.

In brief, the baboons were injected with indium-111-labeled autologous platelets (1 mCi) before thecontrol experiment and with iodine-125-labeledfibrinogen (FGN) (0.005 mCi) before each experi-ment. At the time designated 0 minutes, the shuntswere extended with a 5-cm-long, 4-mm-i.d. Dacrongraft segment. Thrombus formation in these graftswas observed both in real time and retrospectively.The real-time observations involved radioimaging of"1In-labeled platelet deposition with a scintillationcamera and concurrent measurement of the shuntblood flow rate using a Doppler flowmeter. Increasedplatelet deposition and reduced blood flow werepositive signs of thrombus formation. Because depo-sition of platelets from circulating blood is directlyrelated to exposure time and circulating plateletcount, the inhibition of platelet deposition was nor-malized with respect to control values as describedelsewhere.18 The grafts were removed if occlusionoccurred before 60 minutes from the beginning of theexperiment and were washed and stored in 2.5%glutaraldehyde. 125I-labeled FGN radioactivity wasmeasured in counts per minute (cpm) after allowingthe graft "'In activity to decay for at least 30 days. At30 days, the remaining platelet-bound "`In radioac-tivity was 0.006% of the value at 0 minutes, comparedwith the 70% of `2I radioactivity that remained at 30days. The remaining `'In radioactivity was sub-tracted from the total radioactivity and total fibrindeposition was calculated by dividing this value bythe plasma radioactivity determined at the same timeas the graft radioactivity (cpm/ml) and multiplying bythe circulating FGN concentration (mg/ml).The animals were physically examined and care-

fully observed for signs of spontaneous bleeding,including determinations of hematocrit and hemoglo-bin before and after each experiment. Standardizedtemplate bleeding times were performed before andduring rAPC infusion to judge the effect of APC onprimary hemostasis.

Simultaneously with the initiation of blood flow,rAPC was infused proximal to the graft at rates ofeither 0.25 or 1.0 mg/kg-hr, as described in earlierexperiments.15 One third of the enzyme dose wasgiven as a bolus and the remaining two thirds weregiven by continuous infusion. On the first day of thestudy, each animal had a control infusion of saline,and on each of two subsequent days, each receivedone lower and one higher dose of rAPC.

The rAPC was activated from recombinant proteinC (rPC) using immobilized thrombomodulin-throm-bin. The preparation, purification, and characteriza-tion of rAPC has been described elsewhere.18The specific activity of APC was determined in a

clotting assay.'5 Briefly, the assay was based on theability of APC to prolong the activated partial throm-boplastin time (APTT). Normal human plasma(NHP) or rAPC was diluted and incubated inProtac®, American Diagnostica Inc., Greenwich,Conn., a snake-venom activator of PC. PC-depletedhuman plasma and APTT reagent were then addedand the mixture was clotted by recalcification. Thespecific activity of APC was determined by compari-son of results using standard dilutions of the NHPand of the enzyme.The anticoagulant activity of the circulating rAPC

was determined from citrated plasma samples in anAPYT assay using a fibrometer. The assay was per-formed within 5 minutes of blood sampling to mini-mize in vitro inhibition of the enzyme by its physio-logical plasma protease inhibitors. Standard dilutionsof rAPC were made in vitro using a preinfusionplasma sample from each animal, with the resultsgiven as a standard reference curve for each animal.Circulating levels of procoagulant factors VIII and V(FVIII and FV) were measured with factor deficienthuman plasma (George King Biomedical, Inc., Over-land Park, Kan.) after inhibition of the anticoagulantrAPC in the samples. The rAPC in 10 ,ul citratedbaboon plasma was inhibited by addition of 20 ,ulimmunoaffinity-purified polyclonal sheep anti-humanPC antibodies (aPC-pab, 3 ,uM final). The aPC-pabwas purified from sera of immunized sheep (kindlyprovided by Dr. H.P. Schwarz, Vienna, Austria). Thespecific IgG from the IgG fraction was absorbed to aSepharose column containing immobilized humanPC, eluted with 4 M guanidine, and dialyzed intoTris-buffered saline. After incubation of the sampleswith aPC-pab for 460 seconds at 370 C in Tris-buffered saline (pH 7.4) containing 0.5% casein in afinal volume of 40 gl, 100 1.l APTT reagent andfactor-deficient human plasma were added and incu-bated for 200 seconds, and the clotting time wasdetermined after recalcification with 100 ,u1 CaCl2 (25mM). Pooled normal baboon plasma (NBP, 10 ul)incubated with rAPC (10 gl, 60 nM) and aPC-pab(20 pA) was used as a reference standard. TheaPC-pab abolished the anticoagulant activity of 60nM APC added to NBP or to the 0-minute baboonsamples. The APTT was longer than 2 minutes withrAPC and without aPC-pab, 52.3 +3.8 seconds (n=9)with both rAPC and aPC-pab, and 50.5 ±4.3 seconds(n=7) without APC and with aPC-pab. The FV andFVIII levels were determined as percentages ofcontrol values using the NBP standard curve.The amidolytic activity of the circulating rAPC and

the circulating levels of the enzyme were determinedin a solid phase assay as described.15 Briefly, theenzyme in plasma was protected from its physiologi-cal inhibitors with citrate/benzamidine acting as anti-



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FIGURE 1. Plots ofchanges in circulating activatedrecombinant protein C (rAPC) plasma levels duringand after 60 minutes infusion. rAPC was adminis-tered at two doses- 0.25 mg/kg-hr (panel A) and1.0 mg/kg-hr (panel B)-into baboons, and mea-surements ofcirculating levels were detennined usingamidolytic (0-*) and anticoagulant (A- - -A)assays. Values are given in micrograms per milliliteras mean±SEM as a function oftime fiom beginningof infusion.

15 60 75 90 15 60 75 90

Time in minutes

coagulants. It was separated from plasma using animmobilized monoclonal anti-human PC light-chainantibody (designated C3), and the enzymatic activitybound to the immobilized antibody was measuredusing a chromogenic substrate.The effect of rAPC on in vitro clot lysis was

determined by measuring radiolabeled FGN andfibrin degradation products released into the sera ofwhole blood clots formed ex vivo. Immediately afterdrawing 0- and 45-minute arterial blood samples (0.5ml) from baboons receiving rAPC infusion, the bloodwas mixed and clotted in glass tubes containing 10 glhuman thrombin (1 lU/mi final concentration) withor without 10 ,ul tissue-type plasminogen activator(t-PA; 15 ng/ml final concentration; t-PA kindlyprovided by Dr. D. Collen, University of Leuven,Belgium). After incubation for 20 hours at 370 C toallow coagulation and fibrinolysis to proceed, theclots were centrifuged (3,000g for 10 minutes). Onehundred microliters of supernatant (sera) was sepa-rated and both the sera and the pellet (clot plusresidual sera) were stored for 30 days, allowing99.994% of the pellet 1"'in radioactivity to decaybefore determination of '25I radioactivity in a gammacounter (model 4/600, Micromedic Systems, Hor-sham, Pa.). An increase in the percentage of nonsed-imenting radioactivity (in sera) compared with totalradioactivity (in whole blood sample) following rAPCinfusion (45 -minute sample compared with the 0-minute sample) was an indicator of the enhancementof whole blood clot lysis due to rAPC.

Indirect markers of thrombus formation (e.g.,FGN, fibrinopeptide A [FPA], /-thromboglobulin[,BTG], platelet factor 4 [PF4], and D-dimer levels inplasma samples) as well as measurements of bleedingtime, circulating platelet count (PLC), hemoglobinconcentration (HB), and hematocrit (HT) were de-termined as described elsewhere.16,17,19

Statistical analyses of the results were performedusing either the Mann-Whitney U test withoutassumptions about the normality of the populationdistribution or by analysis of variance (Studentizedrange test) when more than two means were

compared,20 unless otherwise stated. Values are givenas mean+SEM unless otherwise stated.

ResultsCirculating Levels ofActivated RecombinantProtein CTo measure the rAPC activity, blood samples were

drawn at 0, 15, 60, 75, and 90 minutes after initiationof blood flow through the Dacron graft. The infusionofrAPC started at 0 minutes and was terminated at 60minutes. Figure 1 (panels A and B) illustrates theplasma levels of the enzyme calculated from theresults of the clotting and amidolytic assays whenrAPC was infused at two different doses. The level ofcirculating endogenous APC that might have beengenerated during control experiments was below thedetection limit of both assays. In the lower-doseexperiments (Figure 1A) the two activity assays gavesimilar results. rAPC activity in plasma was 0.42+0.05mg/l (amidolytic assay) or 0.39±0.06 mg/l (anticoag-ulant assay) at 15 minutes and 0.34±0.04 mg/l (ami-dolytic) or 0.49±0.17 mg/l (anticoagulant) after 60minutes of infusion. Circulating rAPC activitydecreased to one half of the 60-minute value (amido-lytic) by 8-10 minutes after termination of the infu-sion. During infusion of 1.0 mg/kg-hr rAPC (Figure1B) the APTT values increased from 38 seconds tomore than 2 minutes. Therefore, the plasma levels ofrAPC were calculated from the amidolytic activityonly. Circulating rAPC levels of 2.0+0.2 mg/l at 15minutes and of 1.9±0.2 mg/l at 60 minutes weremeasured. The enzymatic activity of circulating rAPCdecreased by 50% in 12 minutes after the infusion wasterminated at 60 minutes. The difference between therAPC levels of the samples from the higher- andlower-dose infusion experiments was statistically sig-nificant (p<0.01, n=8 in each group).

Antithrombotic EffectsGraft patency. Blood flow rates averaged 195 ±46

ml/min (26 cm/sec) through the 4-mm-i.d. graft seg-ment. This rapid arterial flow resulted in the forma-

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Ir

X

v

0

0 15 30 45 60Time In minutes

FIGURE 2. Plot ofpatency ofarteriovenous Dacron vasculargrafts throughout 60 minutes of control experiments (o - o)and during infusion of 0.25 (A-A) and 1.0 mg/kg-hr rAPC(n-*). Results are given as percent open. Proportion ofopengrafts is also given at 15, 30, 45, and 60 minutes.

tion of platelet-rich thrombi, as shown previously byscanning electron microscopy.16 This process gener-ally results in graft occlusion. Figure 2 summarizesthe measurements of graft patency. Five of eightcontrol grafts occluded within 60 minutes when norAPC was infused. In 16 infusions of either the loweror the higher dose of rAPC, none of the graftsoccluded, and blood flow rates did not change overthe 60 minute study interval.

Platelet deposition. Platelet deposition measure-ments determined from real-time imaging are shownin Figure 3 (panel A). The steady increase in plateletdeposition observed with all control grafts was inter-rupted by thrombotic occlusion in five grafts. Thethree control grafts which were patent at 60 minutescontained an average of 13.5±1.2x 109 depositedplatelets. All eight grafts in the experiments with thelower dose of rAPC were patent at 60 minutes andcontained 10.2±1.5 X 109 deposited platelets. Plateletthrombus accumulation was further reduced to4.1 ±0.6x 109 platelets when 1.0 mg/kg-hr rAPC wasinfused, a value that was significantly less than thatseen after the infusion of the 0.25 mg/kg-hr dose(p<0.02). At 30 minutes, platelet deposition wasinhibited, compared to control values, by 12.8±9.6%(n=7, p>O.O5) when rAPC was infused at the lowerdose and by 42.3±13.2% (n=7, p<0.01) when thehigher dose was given. Inhibition of platelet deposi-tion was not calculated in one baboon because thecontrol graft occluded at 25 minutes.

Fibrin deposition. The effect of rAPC infusion ontotal fibrin deposition was determined in six animals.Figure 3B summarizes the results calculated from themeasurements of 1251-related graft radioactivity. Theaverage termination time of those experiments was at43 minutes due to thrombotic occlusions in four ofthe six control studies. A mean of 7.09± 0.78 mg fibrinaccumulated in the six control grafts. When 0.25mg/kg-hr rAPC was infused into six animals, fibrindeposition averaged 4.05±0.50 mg after 60 minutes.After infusion of 1.0 mg/kg-hr rAPC for 60 minutes,2.8±0.44 mg fibrin was contained in the grafts.Analysis of variance (one-way, p<0.05) and rank test

1

I.

io B 0

100 0

0

4 0.25 CD-1.0 >J

2 m_*n

0I20 4 00 20 40 60

Time in minutes

FIGURE 3. Plots of changes in platelet and fibrin depositionin Dacron vascular grafts. During controls (C, o- - -o) and60 minutes of infusion of either 0.25 (A -A) or 1.0 mg/kg-hr(n-*) rAPC into baboons, platelet deposition (panel A) andfibrin accumulation (panel B) were measured. Results inpanel A were derived from real-time radioimaging of 111In-labeled platelet deposition and displayed as mean+±SEM.Number ofpatent controls (N) is given at different timepoints,indicating thrombotic occlusions throughout the 60-minuteperiod. Results in panel B are derived from analysis of endpoint 125'-labeled fibrinogen deposition determinations. Allvalues are given, and means for different doses are indicatedby horizontal arrows and bars.

(two-tailed,p<0.01) indicated that the three sampleswere significantly different from each other. No validcomparison could be made between the control andrAPC treatment experiments because of the earlyocclusions in controls.

Circulating markers of thrombus fornation andthrombolysis. Table 1 summarizes the results of deter-minations of plasma levels of FPA, BTG, and PF4.Levels of circulating markers of fibrin formation(FPA) and platelet activation (PTG, PF4) weresignificantly increased at the end of the controlexperiments. Infusion of 0.25 mg/kg-hr rAPC pre-vented the increase in FPA levels but only modestlyreduced the elevation in levels of PF4 and f3TG; thatis, the lower dose of rAPC infusion did not preventthe increase in circulating markers of platelet activa-tion. However, after infusion of 1.0 mg/kg-hr rAPCno significant elevation of FPA, PF4, or B3TG levelscould be detected. D-Dimer levels were not elevatedsignificantly after 45 minutes of saline infusion incontrols and did not change after rAPC infusioncompared with the control or preinfusion values. The

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TABLE 1. Changes in Circulating Markers of Thrombosis in Baboons Infused With Activated Recombinant Protein C

Dose of rAPC (mg/kg-hr)

Marker Sample 0 0.25 1.0

PLT (x109/l) Pre 310±40 380+55 360+45Post 240+33 p<0.05 365±43 p<O.OS 370+50

p<0.05 p>0.05 p>0.05PF4 (/tLg/l) Pre 5.1±3.2 8.3±3.5 6.9±2.8

Post 22.0+15.6 p>0.05 14.8±5.5 p<0.05 9.3±1.6p<0.02 p<0.05 p>0.05

,BTG (.g/l) Pre 4.0±2.7 4.6+2.4 4.6+5.9Post 27.3±8.6 p<0.05 12.5±5.4 p<0.02 6.6±3.0

p<0.02 p<0.05 p>0.05FGN (g/l) Pre 3.9±1.1 2.7±0.3 3.3±0.7

Post 3.7±1.5 p>0.05 2.6±0.4 p>0.05 3.3+0.6p>0.05 p>0.05 p>0.05

FPA (pM) Pre 3.9±0.9 4.5±1.7 4.5±2.6 n=5Post 18.0±6.0 p<0.05 4.6±1.4 p<0.05 3.9±1.8 n=5

p<0.05 p>0.05 p>0.05D-Dimer(ng/l) Pre 320±130 260±150 450+240

Post 430±140 p>0.05 270±160 p>0.05 420±290p>0.05 p>0.05 p>0.05

rAPC, activated recombinant protein C; PLT, circulating platelet count; PF4, platelet factor 4; ,BTG, 13-thromboglobulin; FGN, fibrinogen; FPA, fibrinopeptide A.

Values are given as mean±SEM of results determined from plasma or blood samples taken at 0 minutes (pre) andafter 45 minutes of rAPC infusion, or earlier if occlusion had occurred before 45 minutes, as in some controls (post).The probability values (p) for the difference between the pre and post data are given for each marker within eachcolumn below the mean values for pre and post results. Comparisons (p) of the peak values for each marker at 0.25 or1.0 mg/kg-hr (second and third columns) to the peak value in the control study with no rAPC (first column) aredisplayed on horizontal lines.

difference between the percent change in the D-dimer level after the higher-dose experiments (-8%)and that after the control infusion (15%) was notstatistically significant (p>0.05).

In Vivo Parameters of HemostasisBleeding time. Preinfusion bleeding times averaged

4.5+±1.2 minutes (n=16). Plasma levels of circulatingrAPC and measurements of bleeding time (marker ofprimary hemostasis) were not significantly correlated,as neither the lower nor the higher dose of rAPCprolonged the bleeding time significantly (5.2±1.3 min-utes and 5.1+± 1.5 minutes, respectively [n=8]). Norebleeding was observed within 30-40 minutes fromthe standardized cutaneous cuts made between 15 and30 minutes after initiation of rAPC infusion. There wasno sign of extravasation of blood during rAPC infusion;that is, no ecchymoses or hematomas appeared onvisible areas of the body and there was no oozing fromsmall cuts. A minimal but significant drop in PLC wasobserved shortly after completion of control experi-ments; however, there was no change in PLC whenrAPC was infused during perfusion of the graft. Therewas no significant acute fibrinogen consumption duringthe perfusion of the grafts (Table 1). HB and HT valuesdid not change significantly in the control and rAPCinfusion experiments. The animals' pulse rates and theblood flow in the grafts remained stable.

In Vitro Parameters of HemostasisClotting time. Infusion of rAPC caused a prolonga-

tion of the clotting time, a marker of secondaryhemostasis. The APTT increased from 36±1 secondsto 75±5 seconds in the lower-dose experiments andfrom 36±2 seconds to 160±18 seconds in the higher-dose experiments after 15 minutes of rAPC infusion.The corresponding values at the end of the 60-minute infusions were 85±7 seconds and more than180 seconds. FV activity was decreased by 2.4± 13.4%at the end of the experiments with the lower dose ofrAPC and by 14.6±6.1% at the end of the experi-ments with the higher dose. The corresponding val-ues for FVIII activity were decreased by 12.4±11.1%and 11.5+14.5%, respectively. The decrease in theprocoagulant activity of circulating FV and FVIIIwas not statistically significant (p>0.05, n=7 for allmeasurements).

Profibrinolytic effect. t-PA at a low concentration of15 ng/ml incorporated into whole blood clots in vitroinduced a nonsignificant increase in fibrinolysis asmeasured by detection of 15I-FGN-related radioactiv-ity in the sera after 20 hours of incubation (Table 2;preinfusion samples). Infusion of 0.25 mg/kg-hr rAPChad no effect on the in vitro fibrinolysis under theseassay conditions. No increase in fibrinolysis followingrAPC infusion could be detected when the clot did not



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TABLE 2. Effect of Infused Activated Recombinant Protein C onWhole Blood Clot Lysis Induced by Tissue-Type PlasminogenActivator

Dose of rAPC(mg/kg-hr)

0.25 1.00

Radioactivity in sera (%)t-PA added Sample of whole blood

None Pre 40±17 35±19Post 35±13 33±14

p>0.05 p>0.0515 ng/ml Pre 43±18 40±22

Post 42±16 70±32p>O.OS p<O.OS

rAPC, activated recombinant protein C; t-PA, tissue-type plas-minogen activator.

Values represent soluble `2I-fibrin radioactivity in sera as apercentage of the radioactivity in the whole blood sample after24-hour incubation. n=4 for each group. Blood was drawn frombaboons at 0 minutes (pre) and at 45 minutes (post) after initiationof rAPC infusion and clotted using 1 unit/ml thrombin± 15 ng/mlt-PA (final concentrations). Pre and post results were comparedstatistically to give indicated p values.

contain exogenous t-PA. However, when the clot wasprepared from blood samples taken after infusion of1.0 mg/kg-hr rAPC and the tubes also contained t-PA,there was a significant increase in the clot supematantradioactivity (from 40±11% in the 0-minute sample to75 ±18% at 45 minutes,p<0.02), suggesting that someenhanced clot lysis occurred.

DiscussionBecause plasma-derived APC was previously shown

to be a potent antithrombotic agent at plasma levelsthat did not compromise primary hemostasis,18 theantithrombotic activity of rAPC was studied with thesame experimental model of platelet-dependent arte-rial thrombosis. Infusion of rAPC gave plasma levelsof rAPC similar to those of the plasma-derived mate-rial seen in our previous experiments. The circulatinghalf-life of rAPC after continuous infusion wasstopped was somewhat shorter (8-12 minutes) thanthat of plasma-derived APC (12-15 minutes). Thisfinding may indicate minor differences in the inhibi-tion or clearance of plasma-derived APC comparedwith the genetically engineered material.The APTT was a useful indicator of the circulating

level of the enzyme when measured immediately,that is, within 2-5 minutes after blood drawing;however, the accuracy of the assay was limited athigher rAPC plasma levels due to prolongation of theclotting time to more than 2 minutes. Another limi-tation of the clotting assay for APC activity was itslack of specificity. The APC amidolytic assay was veryspecific and more sensitive than the APTI by at leastone order of magnitude. Thus, the APTT assay wasused as a complementary assay for determination ofthe level of the circulating enzyme. Because the

and useful for rapid determinations of circulatingrAPC at plasma levels lower than 1 mg/l.The observation that none of the grafts occluded

during rAPC infusion provided evidence that thehuman recombinant enzyme was antithrombotic.Because plasma levels of 0.4 mg/l rAPC preventedthrombotic occlusion, the qualitative method of sim-ply measuring blood flow was of no use for quanti-tating the antithrombotic effect of rAPC. Using thisqualitative method, rAPC was previously shown byothers21 to inhibit the thrombotic occlusion of exper-imental arteriovenous shunts in baboons.

Information regarding dose-response effects wasobtained by dynamic measurements of platelet andfibrin thrombus formation. Inhibition of platelet dep-osition was evident at 30 minutes in experiments withthe higher dose of rAPC. However, the antithrom-botic effect of rAPC in the lower-dose rAPC infusionexperiments was not evident at 30 minutes. Theinhibition of platelet deposition could not be deter-mined in experiments carried out to 60 minutes dueto progressive occlusion of five control grafts. Thedose-dependency results at 30 minutes were some-what different from our previous experience withplasma-derived APC,15 in which APC had a morepronounced inhibitory effect on platelet deposition(43% and 76% inhibition for the 0.25 and 1.1 mg/kg-hr APC infusion experiments, respectively). Thisdifference between rAPC and plasma-derived APCwas statistically significant (p<0.03 for the lowerdose andp<0.05 for the higher dose). Because rAPCwas at least as effective an anticoagulant as plasma-derived APC in the APTT assay, no obvious expla-nation exists for this difference in antithromboticeffects.The fibrin content of the graft thrombus at the end

of the experiment was negatively correlated with theplasma level of rAPC (r, -0.89); the mean fibrindeposition values after infusion of 0.25 and 1.0mg/kg-hr rAPC for 60 minutes were lower than thecontrol values. Although the two means of fibrindeposition values were slightly different in the lowerand in the higher rAPC infusion dose experiments,suggesting dose-dependency for the antifibrin effect,they did not reflect the difference in the circulatingrAPC levels. Because these results represent endpoint determinations, the time course of inhibitioncould not be quantitated as in the case of plateletdeposition. Establishing the dose-dependency mightrequire several dose range studies.Plasma markers of fibrin formation (FPA), platelet

activation (,3TG and PF4), and fibrinolysis (D-dimer)remained at baseline levels after infusion of 1.0mg/kg-hr rAPC. Because FPA, PTG, and PF4 levelsincreased during control experiments, these datafurther document the potent antithrombotic activityof rAPC in this model. Procoagulant FV and FVIIIlevels did not decrease significantly during control orrAPC infusion experiments, suggesting that circulat-ing rAPC, at plasma levels of up to 2 ,ug/ml for 60APTT could be measured quickly, it proved reliable



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minutes, does not deplete FV or FVIII or produce anacquired coagulation factor deficiency.As observed in studies with plasma-derived APC,15

rAPC had no effect on the bleeding time, even at aplasma level of 1.9 mg/I. This finding is remarkablebecause most agents that are antithrombotic in thisthrombosis model also impair primary hemostasis, asreflected by prolonged bleeding times. These agentsinclude a synthetic peptide-chloromethyl ketoneinhibitor of thrombin (PPACK),17 hirudin,22 mono-clonal antibodies against platelet glycoprotein IIb/I11a,19 and some clinically used antiplatelet drugs.23The mechanism of the antithrombotic effect of

APC in the platelet-dependent thrombosis model isnot completely clarified. APC has no documentedantiplatelet activity in vitro; for example, it does notinhibit platelet aggregation or adhesion. AlthoughAPC acts as an anticoagulant by preventing thrombingeneration, therapeutic doses of another antithrom-bin anticoagulant (heparin, 100 units/kg) do notinhibit platelet deposition.24 Hirudin, the most spe-cific known inhibitor of thrombin was, however,shown to be a potent antithrombotic agent in thesame model.22 Therefore, we think that the antifibrinand antiplatelet effects of APC are related to itsinhibition of thrombin generation and that plateletdeposition in this model of arterial thrombosis is athrombin-dependent process.The ex vivo experiments on the profibrinolytic

effect of rAPC were performed to examine whetherrAPC could enhance the profibrinolytic potential oft-PA in this primate, because enhancement offibrinolysis could contribute to the antithromboticeffect of the enzyme. Incorporation of 1.9 mg/lcirculating rAPC into ex vivo-formed blood clotspromoted t-PA-induced fibrinolysis. Assuming thatcirculating rAPC was also incorporated into graftthrombi, enhancement of fibrinolysis might haveoccurred in vivo as well, in accord with the results ofother investigators.2526 However, clot lysis in vitrowas not increased in the absence of exogenous t-PA,and the D-dimer levels during infusion of bothplasma-derived APC15 and rAPC did not increase,failing to demonstrate induction of fibrinolysis as aresult of APC infusion. These data, however, do notexclude the possibility of enhancement of fibrinolysis,because fibrin generation is inhibited in the presenceof APC. Thus, the suggested in vivo prothrombolyticeffect of APC, which has been based on in vitro or exvivo studies, remains unproven.

In conclusion, rAPC, like plasma-derived APC, wasfound to be an antithrombotic agent in a nonhumanprimate model of arterial thrombosis. The importanceof this observation was enhanced by the fact thatrAPC, like APC, did not impair primary hemostasis asreflected in bleeding time measurements at anti-thrombotic plasma levels, despite its profound effecton the coagulation time in vitro. Moreover, recentstudies showed that infusion of urokinase combinedwith APC in our thrombosis model resulted in a netadditive effect of the antithrombotic activities of each

agent.27 Thus, infused rAPC is a candidate for imme-diate and transient inhibition of arterial thromboticevents, for example, clot formation during cardiacsurgery or rethrombosis after thrombolytic therapy,percutaneous transluminal coronary angioplasty,or endarterectomy.

AcknowledgmentWe gratefully acknowledge Ulla Marzec for invalu-

able technical assistance.

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KEY WORDS * platelets * grafts * fibrinolysis



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A Gruber, S R Hanson, A B Kelly, B S Yan, N Bang, J H Griffin and L A Harkerof arterial thrombosis.

Inhibition of thrombus formation by activated recombinant protein C in a primate model

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