SPECIAl. ARTICLES Research Forums I and II and Breakfast Session articles presented at the Joint Annual Meetings of the SVS/ISCVS, June 2-5, 1991.

Future alternatives to heparin: Low-molecular-weight heparin and hirudin Cattos E. Donayre, MD, Kenneth Ouriel, MD, Robert Y. Rhee, MD, and Cynthia K. Shortell, MD, Rochester, N.Y. The antithrombotic effects of standard heparin were compared with those of lowmolecular-weight heparin (LMWH) and hirudin by use of an in vitro perfusion system. Fresh blood collected from human volunteers was treated with varying doses of these three agents and perfiased in a recirculating system over everted porcine vein segments. A low shear rate (100/sec) was selected to simulate conditions in large arteries and veins. Platelet and fibrinogen deposition were evaluated with indium 111 and iodine 125 radiolabels, respectively. Anticoagulant activity was assessed by measuring the activated dotting time (ACT). Anti-Xa activity was assayed to determine the degree to which these agents used antithrombin III pathways. Low-molecular-weight heparin was the weakest anticoagulant, requiring 32 ~g/ml blood to double the ACT. By contrast, the ACT doubled with only 0.75 and 1.10 isg/ml blood ofheparin and hirudin, respectively. Heparin and hirudin inhibited platelet and fibrin deposition at equivalent doses. Low-molecular-weight heparin was a less potent inhibitor of fibrin than heparin or hirudin. Hirudin, a direct thrombin inhibitor, exhibited minimal anti-Xa activity, contrasted with 0.14 anti-Xa units/l~g for LMWH and 0.13 anti-Xa units/mg for heparin. These data suggest that heparin and hirudin are more potent anticoagulants and antiplatelet agents than LMWH. (J VASC

SURG 1992;15:675-82.)

Heparin is the most commonly used parenteral anticoagulant. It is inexpensive, effective, and readily reversible with protamine sulfate, but it is associated with a variety of undesirable side effects including bleeding, platelet aggregation, and thrombocytopenia. The search for anticoagulants with modified biologic actMties, improved pharmacologic properties, and lower complication rates led to the development of low-molecular-weight heparin (LMWH) and rekindled an interest in hirudln, the oldest known anticoagulant. The present study was undertaken to From the University of Rochester School of Medicine and Dentistry Department of Surgery (Dr. Rhee) Division of Vascular Surgery (Drs. Donayre, Ouriel, Shortell), Rochester. Supported by NIH/PHS grant HL40889. Presented at the Forty-fifth Annual Meeting of the Society for Vascular Surgery, Research Forum I, Boston, Mass., June 4-5, i991.

Reprint requests: Kenneth Ouriel, MD, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642-8410. 24/1/34392

compare the antiplatelet arid anticoagulant properties of standard heparin, LMWH, and hirudin with use of an in vitro whole blood perfusion system. It is hoped that the results of this study will serve to elucidate the anticoagulant potential of these agents. METHODS Fresh blood from normal human volunteers was collected by use of standard tourniquet technique, steel needles, and plastic syringes. The blood was not stored or citrated and was introduced in the perfusion system within 60 seconds after withdrawal. Patients with a history of aspirin or other nonsteroidal antiinflammatory drug use and those taking oral contraceptives were excluded from the study. Porcine vein segments were obtained from a local slaughterhouse (Conti Packing Co., Inc., Rochester, N.Y.). Internal mammary veins measuring 5 mm in diameter were harvested from freshly slaughtered pigs and sectioned to 2 cm in length. All veins were stored in cold 0.1 mol/L Sorensen's buffer solution (pH 7.2). 675

Journal o f VASCULAR SURGER'£ ~

676 Donayre et al.

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Fig. 1. Clear acrylic annular perfusion chamber with everted deendothelialized subdavian vein. Endothelial injury was induced by air sufflation at 1 L/min for 10 minutes. 1 The venous segments were then everted, mounted on clear acrylic rods, and placed in an annular perfusion chamber2 (Fig. 1).

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Radioactive labeling o f platelets and fibrinogen Fresh human donor blood was used for autologous platelet labeling. The average labeling efficiency (platelets with incorporated radioactivity/platelet activity + supernatant activity × 100) was 69%. Platelet deposition was quantitated as platelets/cm2 of venous surface. Conversion of raw counts/min to platelets/cm2 was accomplished as follows3. Platelet deposition = (Vesselm In counts) x (Perf-usateplatelet count) (Peffusaten~ In counts) x (Vessel surface area) Commercially available radiolabeled human fibrinbrinogen (iodine 125 [1251], Ibrin Amersham Corp., Arlington Heights, Ill.) was used. Fibrinogen concentration in the blood was assayed spectrophotometrically.4 Fibrin deposition (txg/cm2) was assessed as follows: Fibrin deposition = (VesseP2s I counts) x (Perfusate fibrinogen cone.) (Perfusate1/s I counts) x (Vessel surface area) The presence of radiolabeled platelets and fibrin was assessed with a two-channel gamma counter (Nuclear Chicago 1195 Automatic Gamma System, Des Plaines, Ill.). Indium 111 (nlIn) activity was assayed immediately after perfusion; correction for crossover from the 125I channel was made by use of standard calculations. Iodine 125 activity was assayed by gamma counter 30 days after the completion of the perfusions, after all of the rain had decayed. Once again, standard methods were used to calculate 12sI activity immediately after perfusion.

Activated clotting time (ACT) determination Activated clotting times (International Technidyne Corp., Edison, N.J.) s were obtained immediately after withdrawal of blood from the donor and obtained again after the addition of radiolabeled platelets, fibrinogen, and anticoagulant.

Flowmeter

Fig. 2. Experimental setup with peristaltic pump, flow meter, and perfusion chamber. Anti-Xa determination The LMWH compounds used (RD Heparin no. 13188; Hepar Industries Inc., Franklin, Ohio) had 133 units anti-FXa activity/rag of LMWH and 95 USP units of anticlotting activity/rag of LMWH, corresponding to 1.4 anti-FXa units/USP unit, according to the manufacturer's specification. A chromogenic competing substrate assay (Coatest; KABI Diagnostics, Franklin, Ohio) was used to determine the anti-Xa activity of heparin and hirudin. 6

Vessel perfusion The radiolabeled whole blood was circulated across everted venous segments in the annular perfusion chamber (Fig. 2) for 3 minutes at a shear: of 100/see. Vessels were removed from the internal rod at the completion of the perfusion and washed with 0.1 mol/L Sorenson's buffer to remove nonadherent blood7

Experimental groups (1) Control: saline alone; no anticoagulant added. (2) Standard porcine intestinal heparin sodium solution (1000 USP units/mg, Elkins-Sinn Inc., Cherry Hill, N.J.) was diluted with normal saline to 100 USP units/ml and added in six doses: 0.038, 0.075, 0.19, 0.375, 0.75, and 1.50 Ixg/ml blood. (3) LMWH (RD Heparin: RD no. 13188, Hepar Industries) 95 USP units/mg was diluted with normal saline solution to 100 USP units/ml and added in six doses: 0.79, 2.00, 3.95, 7.90, 15.79, and 31.58 ~g/ml. (4) Hirudin (recombinant hirudin no. HBW025, Hoechst-Roussel Pharmaceuticals Inc.,

Volume 15 Number 4 April 1992

Alternatives to heparim L M W H and hirudin

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Fig. 3. Effects of increasing doses of heparin, LMWH, and hirudin on the ACT with use of a log scale. Table I. Comparison of heparin, hirudin, and L M W H on ACT, platelet and fibrin deposition with iDto o (dose required to double the ACT), EDso (dose required to achieve 50% inhibition of control value), and maximum inhibition achieved ACT

Fibrin Deposition

Hatelet deposition

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El)so

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Max inhb (%)

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1.50 31.58 3.00

74.2 65.4 89.9

0.075 3.95 0.25

0.75 31.58 3.00

88.0 87.7 90.0

Table II. Platelet and fibrin inhibition with increasing doses of heparin; effects on ACT Heparin No.

Dose (#g/ml)

USP (units/ml)

8 4 4 4 4 4

Control 0.075 0.19 0.38 0.75 1.50

0 0.075 0.19 0.38 0.75 1.50

A C T (see) 117 132 158 170 240 273

-+ 10 ± 9 +- 5 ± 21 ± 8** ± 35*

Fibrin deposition (#g/cm 2) 95.7 85.0 62.8 45.3 38.3 24.8

-± 23.3 --- 2.8 ± 10.3 ± 8.7 ± 17.9 ± 5.3*

Platelet deposition (million/cm2) 39.9 18.6 8.3 8.5 4.8 5.4

- 10.7 -+ 9.1 ± 0.8* -+ 2.1 ± 1.0" ± 1.4

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~p < 0.01 Somerville, N.J.) 1 mg diluted in 10 ml of normal saline and added in five doses: 0.25, 0.50, 1.00, 2.00, and 3.00 g g/ml blood. Equivalent volumes of saline were added to the perfusate in each group to eliminate differences as a result of dilution. D a t a analysis

The differences between groups were evaluated by use of analysis of variance (ANOVA) and the Student's t test. Differences were considered significant whenp < 0.05. RESULTS Activated clotting time Anticoagulant activity was assessed by measuring the ACT (Fig. 1, Table I). A strong correlation was

observed between heparin dose and ACT (Table II, F = 12.13,p < 0.001). The EDlo 0 for heparin (the dose required to double the ACT) was 0.75 ~xg/ml blood. Hirudin, like heparin, was associated with a marked increase in the ACT at low doses (Fig. 3; Table III, F = 5.36,p < 0.0) with an ED~o o of i ~g hirudin/ml blood. Compared with heparin and hirudin, L M W H was a less potent anticoagulant requiring much larger doses to alter the ACT. Significant prolongation of the ACT occurred only after a dose of 7.90 ~g LMWH/ml blood (Table IV), and the EDmo was 31.58 Ixg LMWH/ml blood. Fibrin deposition The amount of 125Ilabeled fibrin deposited on the vessel wall decreased with increasing heparin concen-

Journal of VASCULAR SURGERY

678 Donayre ¢t al.

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Table III. Platelet and fibrin inhibition with increasing doses of hirudin; effects on ACT Hirudin No.

Dose Ozg/cc)

8 4 4 4 4 4

Control 0.25 0.50 1.00 2.00 3.00

Fibrin deposition Ozg/cm2)

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± ± ± ± ± ±

10 6* 18" 15" 21"* 19"*

Platelet deposition (million/era2) 39.9 +_ 10.7

95.7 26.8 37.0 37.0

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Dose (tag/ml)

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Fibrin deposition (~g/cm 2) 95.7 58.1 28.5 36.6 37.1 33.8 33.3

-+ 23.3 ± 8.3 +_ 2.3 ± 2.1 ± 5.5 ± 4.0* ± 3.0*

Platelet deposition (million/cm 2) 39.9 24.3 18.9 19.3 11.7 10.7 4.9

-4- 10.7 ± 10.7 ± 6.7 ± 3.7 ± 2.9 ± 0.8 -+ 1.7"

*p < 0.5 **p < 0.01

trations (F = 4.81, p < 0.003) (Fig. 4, Table II). Standard heparin effected a 50% reduction in fibrin deposition (ED5o) at only 0.375 ~g/ml blood, a dose equivalent to 2000 USP units bolus ofheparin in a 70 kg man. Maximum fibrin inhibition (75%) was observed with 1.50 ~g heparin/ml. The ED5o for hirudin was 0.25 tlg/ml blood, and the maximum level of fibrin inhibition (90%) was achieved at a hirudin dose of 3 ~g/ml (Fig. 5, Table III). The effects of L M W H on fibrin deposition are shown in Fig. 6 and Table IV. The EDso was 0.79 ~g LMWH/ml blood, whereas maximum fibrin inhibition (66%) was achieved at 31.58 ~g LMWH/ml

blood. Thus, L M W H had the weakest antifibrin activity of the three agents studied. Platelet deposition Platelet deposition decreased significantly with increasing doses of heparin (F = 13.5, p < 0.001) (Fig. 4, Table II). The EDso of heparin for platelet deposition was only 0.075 ~g/ml blood, equivalent to a 400 USP unit bolus in a 70 kg man. Maximum inhibition of platelet deposition (88%) was achieved with a dose of 0.75 ~g heparin/ml blood. These data show that heparin's antiplatelet effects occur at doses that do not affect the ACT. The ED50 ofhirudin (Fig.

Volume !5 Number 4 April 1992

Alternatives to heparin: L M W H and hirudin

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