Donald E. Macfarlane

Efficacy and Safety of Enoxaparin to Prevent Deep Venous Thrombosis after Hip Replacement Surgery

Enoxaparin is a low-molecular-weight heparin with an average molecular weight of 4500 daltons that is obtained by controlled depolymerization of standard unfractionated porcine intestinal mucosal heparin (average molecular weight, 15 000 daltons) [1]. Enoxaparin and unfractionated heparin inhibit clotting indirectly by accelerating the formation of irreversible complexes between antithrombin III and several activated clotting factors, including factors IIa and Xa [2, 3]. Enoxaparin has several biological properties that differ from those observed in unfractionated heparin. In animal models, prevention of venous thrombosis by equipotent dosage regimens of enoxaparin or unfractionated heparin had equivalent antithrombotic activity. However, reduced hemorrhage was observed with enoxaparin [4, 5]. In in vitro studies, enoxaparin at concentrations with equivalent anti-factor Xa activity had four times less anti-factor IIa activity compared with unfractionated heparin [2, 3, 6]. Furthermore, in contrast to unfractionated heparin, enoxaparin had reduced interactions with platelets and plasma proteins in ex vivo experiments [7, 8]. Enoxaparin also has pharmacologic properties that are advantageous compared with unfractionated heparin. Administered intravenously, the half-life of enoxaparin was four times greater than that of unfractionated heparin (4.4 hours compared with 0.35 hours) [9, 10]. Its bioavailability after subcutaneous injection was more than 90% compared with 29% for unfractionated heparin [10]. These characteristics give enoxaparin potential clinical advantages, including a more predictable dose response and a reduced frequency of administration. Deep venous thrombosis and pulmonary embolism occur frequently in patients having total hip replacement surgery. Deep venous thrombosis has been observed in 40% to 70%, asymptomatic pulmonary embolism in as many as 25%, symptomatic pulmonary embolism in 4% to 19%, and fatal pulmonary embolism in as many as 6.7% of these patients [11-16]. Previous clinical trials showed that it was effective and safe for preventing deep venous thrombosis in patients having total hip replacement when administered after [17, 18] or before surgery [19]. A substantial risk reduction (71%) without an increase in hemorrhage was observed in the incidence of proximal and distal deep venous thrombosis in patients who had total hip replacement and were given 30 mg of enoxaparin subcutaneously after surgery every 12 hours for as long as 14 days, compared with patients who received placebo [17]. Although this study showed the efficacy of enoxaparin administered after surgery, it did not determine an optimum dose or satisfactorily define the drugs safety profile because only one enoxaparin dosing regimen was tested and too few patients were studied. Our dose-ranging study was done to compare the efficacy and safety of three postoperative enoxaparin dose regimens to prevent deep venous thrombosis in patients having total hip replacement surgery. It was designed to identify a clinically effective dose of enoxaparin with a minimal risk for hemorrhagic complications. We compared subcutaneous postoperative administration of 10 mg of enoxaparin once daily, 40 mg of enoxaparin once daily, and 30 mg of enoxaparin every 12 hours. Methods Study Design This multicenter study was conducted as a randomized, double-blind, parallel-group clinical trial, with 32 institutions participating. The protocol and informed consent forms were approved by each centers institutional review board. Patients All patients gave written informed consent before entering the study. Men and women who were 31 years or older and were scheduled for hip replacement surgery, including primary and revision procedures, were eligible. Revision procedures included revision of the acetabular component, the femoral component, or both components. Noninvasive vascular examinations, including strain gauge or impedance plethysmography, Doppler sonography, and duplex or b-mode ultrasonography, were done within 14 days before surgery to provide baseline information and to exclude patients with identifiable deep venous thrombosis. Patients excluded from the study were women with child-bearing potential and patients who had ipsilateral hip surgery within 3 months. Other exclusion criteria included a history of deep venous thrombosis, pulmonary embolism, or both; heparin-associated thrombocytopenia; hemorrhagic disorders; allergy to heparin, protamine sulfate, or radiocontrast agents; eye, spinal cord, or nervous system surgery within 3 months; active ulcerative disease of the alimentary tract; uncontrolled hypertension; or use of nonsteroidal anti-inflammatory agents during the 4 days before surgery. Enrollment began in December 1988 and continued through September 1990. Dosing Schedule The efficacy and safety of three enoxaparin (Rhone-Poulenc Rorer Pharmaceuticals, Collegeville, Pennsylvania) dose schedules were compared: 10 mg once daily, 40 mg once daily, and 30 mg every 12 hours. All doses of study medication were administered by subcutaneous injection. The first dose of study medication was administered within 24 hours after surgery, and the drug was continued for as long as 7 days. Evaluations and Scheduling The primary assessment for deep venous thrombosis was bilateral contrast venography performed on day 7 of treatment, earlier if clinically indicated, or on discharge from the hospital. Institutional physicians who were unaware of patient treatment assignment interpreted venograms and noninvasive vascular examinations. Additional assessments included daily clinical evaluations and noninvasive vascular examinations on treatment days 4 and 7 or as clinically indicated. Clinical evidence of venous thromboembolic disease was defined as documented treatment of clinically evident or symptomatic deep venous thrombosis or pulmonary embolism. Clinical Analysis The primary determinant of efficacy was the incidence of deep venous thrombosis as determined by venography, noninvasive vascular examinations, and clinical evidence. Clinical evidence included reported deep venous thrombosis or pulmonary embolism as an adverse event or the occurrence of symptoms and signs of venous thromboembolic diseases and associated therapy. The primary study conclusions for efficacy and safety outcomes were based on an analysis of all treated patients that included all patients who received at least one dose of study medication [20]. In addition, efficacy was analyzed for evaluable patients, those who received at least 75% of study medication, had adequate bilateral venography, and completed the study with no protocol violation. The primary determinants of safety were the incidence of major and minor hemorrhagic episodes, a decrease in hemoglobin of 20 g/L (2 g/dL) or more compared with the postoperative predose value, hemoglobin values less than 80 g/L (8 g/dL), clinically significant abnormal laboratory results, the number of transfusions required, and the occurrence of serious adverse events. Serious adverse events included death, life-threatening events, and events that resulted in prolonged hospitalization. A major hemorrhagic episode was overt hemorrhage associated with one or more of the following: death or a life-threatening clinical event; acute myocardial infarction or stroke; retroperitoneal or intracranial hemorrhage; postoperative transfusion of more than two units of packed red blood cells (excluding autologous blood); or a decrease of 20 g/dL or more in hemoglobin that was directly attributable to the overt hemorrhagic episode. A minor hemorrhagic episode was an overt hemorrhagic episode that did not meet criteria for classification as major. Interim Analysis Our study was changed from a three-arm to a two-arm trial on 11 May 1990 as a result of a blinded interim analysis conducted in March 1990. The analysis was done on 305 patients and resulted in the discontinuation of a treatment group identified only as A because of the high rate of treatment failure observed in that group. The study blinding was not broken when treatment group A was discontinued. Treatment group A was revealed to be the group receiving 10 mg of enoxaparin when the blinding was formally broken. Because of the smaller number of patients recruited into the group receiving 10 mg, the precision of the estimated incidence of deep venous thrombosis in that group was not as high as that for the other two treatment groups. Statistical Analysis The study population required was determined before initiation and assumed deep venous thrombosis rates of 25% in the group receiving 10 mg of enoxaparin once daily and 12.5% in groups receiving 40 mg once daily and 30 mg every 12 hours. Approximately 195 patients were required per treatment group to achieve 80% power when using a two-tailed test at a significance level of 0.05. Patients were assigned to treatment groups in a 1:1:1 ratio in blocks of six, using a computer-generated randomization schedule. A two-way logistic regression model, including factors for treatment group and center, was used to compare the treatment. Significance testing was done at the 0.025 level based on a Bonferroni correction for the two primary comparisons, 10 mg of enoxaparin once daily compared with 40 mg enoxaparin once daily and 30 mg every 12 hours, respectively [21]. The P values for these comparisons were multiplied by a factor of two to reflect the Bonferroni adjustment. Significance testing between the group receiving 30 mg of enoxaparin every 12 hours and that receiving 40 mg once daily was done at the 0.05 level of significance without Bonferroni adjustment. The incidence of treatment failure was summarized by treatment group in patient subpopulations defined by 1) demographic characteristics [age, sex, and race]; 2) type of surgery [primary or revision arthroplasty]; 3) type of anesthesia; or 4) use of acrylic bone cement and graduated compression stocki

2-Methylthioadenosine[beta-32P]diphosphate. An agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets.

2-Methylthio-ADP and its radioactive analogue [beta-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin E1. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [beta-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was approximately 3.5 X 10(6) M-1 S-1, and the first order dissociation rate was 0.024 s-1, both measured at 23 degrees C. The dissociation equilibrium constant (approximately 15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin E1-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Ki = 3.5 microM), ATP (7 microM), 2-azido-ADP (0.12 microM), inosine diphosphate (IDP, 150 microM), guanosine diphosphate (GDP, 350 microM), and AMP (800 microM). Binding of 2-methylthio-ADP was also blocked by the non-cell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 32 degrees -40 degrees C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was approximately 15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket.

Two dimensional benzyldimethyl-n-hexadecylammonium chloride → sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis: A high capacity high resolution technique for the purification of proteins from complex mixtures

A two dimensional preparative polyacrylamide gel electrophoresis system is described which has both high resolving power and high loading capacity (approximately 100 mg protein). The first dimension is electrophoresis toward the cathode at acid pH in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC), and the second dimension is electrophoresis toward the anode in the presence of sodium dodecyl sulfate. Proteins purified in this way have been sequenced and used successfully as antigens.

Transitioning from Argatroban to Warfarin Therapy in Patients with Heparin-induced Thrombocytopenia

Argatroban, a direct thrombin inhibitor used for thromboprophylaxis or treatment in heparin-induced thrombocytopenia (HIT), is routinely monitored using the activated partial thromboplastin time (aPTT) yet also prolongs the international normalized ratio (INR). Peritransitional INRs, aPTTs, anticoagulant dosing patterns, and outcomes were evaluated in 165 HIT patients who were transitioned, without guidelines, from argatroban to warfarin therapy. Argatroban (median doses: 1.5-2.0 mcg/kg/min) and warfarin (median dose: 5 mg initially with 3.8 mg/day thereafter) overlapped a median 4 days. Median (5-95th percentile) aPTTs (in seconds) and INRs, respectively, were 59.8 (38.8-82.9) and 3.2 (1.7-7.0) during argatroban monotherapy, 68.6 (44.5-104) and 5.3 (2.4-16) maximally during cotherapy, 59.9 (38.7-92.2) and 4.0 (2.2-11.6) immediately before argatroban cessation during cotherapy, and 36.0 (25.6-60.2) and 2.3 (1.3-7.3) within a median 10-12 hours after argatroban cessation. Major bleeding occurred in 1 (0.6%) patient pretransitionally and no patient during or after cotherapy. Eighteen (10.9%) patients experienced 19 peritransitional adverse outcomes (one death, two amputations, 16 new thromboses); these patients had more severe HIT than event-free patients (median baseline platelet count, 39 vs. 83 × 109/L). Of 108 patients with post-transitional INR data, 43 achieved a therapeutic INR (prospectively defined as 1.9-3.5), 34 were subtherapeutic, and 31 were supratherapeutic, with no across-group trend in new thrombosis. Hence in the absence of guidelines, physicians transfer patients from argatroban to warfarin therapy with acceptably low complication rates in HIT, without systematically over- or under-dosing warfarin. Furthermore, INRs greater than 5 commonly occur in HIT patients during argatroban monotherapy and argatroban/warfarin cotherapy, without major bleeding.

Use of benzyldimethyl-n-hexadecylammonium chloride ("16-BAC"), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells.

A discontinuous polyacrylamide gel system operating at pH 4.0-1.5 which resolves proteins bearing base labile groups extracted from intact cells is described. It uses potassium phosphate buffer in the running and stacking gel and glycine as the trailing ion component. Proteins are solubilized with urea and benzyldimethyl-n-hexadecylammonium chloride, a cationic detergent. The utility of the system is illustrated by fluorographs of the pattern of protein methylation in blood platelets and the HL60 promyelocyte cell line.

Structure-activity relationship analysis of substituted 4-quinolinamines, antagonists of immunostimulatory CpG-oligodeoxynucleotides

On the basis of a systematic SAR analysis of substituted quinolines, a derivative 32 was synthesized that shows half-maximal inhibition of the immunostimulatory effect of CpG-oligodeoxynucleotides in vitro at the concentration of 0.24 nM.

Use of [methyl-3H]yohimbine as a radioligand for alpha-2 adrenoreceptors on intact platelets. Comparison with dihydroergocryptine

The binding of the non-selective α-adrenergic receptor antagonist [3H]dihydroergocryptine and the selective α2-adrenergic receptor antagonist [methyl-3H]yohimbine to intact blood platelets was investigated. Loosely bound dihydroergocryptine dissociated from washed platelets diluted with plasma in a temperature dependent fashion, and at 37°C this was substantially complete in 30 seconds. The remaining radioactivity was resolvable into a non-specific component equivalent to about 10μl supernatant per 108 platelets and a saturable component that was suppressed by yohimbine amounting to about 400 sites per platelet. The binding to these sites was slow, and did not equilibrate in 20 minutes. Addition of phentolamine, yohimbine or non-radioactive dihydroergocryptine at this time resulted in the prompt dissociation of about one third of the specifically bound dihydroergocryptine, and a further one third dissociated over the next hour. In contrast, the non-specific binding of yohimbine to intact platelets after plasma dilution amounted to the equivalent of 0.5 − 1.5 μl supernatant per 108 platelets. Specific binding and dissociation was exponential with K1 = 5.25 × 105M−1 sec−1, K−1 = 3.3 × 10−3sec−1. The derived dissociation constant was 6.3 nM, and the rate of dissociation was not materially affected by the presence or absence of non-radioactive yohimbine or epinephrine. Equilibrium binding revealed 200–350 non-interactive sites per platelet with dissociation constant 2–4 nM. Binding was completely suppressed by agents which interact with the α2-receptor with the following rank of potency: epinephrine < clonidine < ρ-aminoclonidine < yohimbinic acid < phentolamine < yohimbone. Dihydroergocryptine and dihydroergotamine also completely suppressed binding. Prazosin, an α1-adrenergic receptor antagonist suppressed binding only at concentrations above about 10μM. It is concluded that dihydroergocryptine appears to bind to more than one site, one of which has such a slow rate of dissociation that equilibrium with low concentrations of dihydroergocryptine will not be achieved for several hours. However, yohimbine bound with the characteristics of a bimolecular reaction with an α2-receptor site, and this reagent is expected to be a useful radioligand for the α2-adrenergic receptor on intact platelets.

P47 phosphoprotein of blood platelets (pleckstrin) is a major target for phorbol ester-induced protein phosphorylation in intact platelets, granulocytes, lymphocytes, monocytes and cultured leukaemic cells: absence of P47 in non-haematopoietic cells

Aggregating agents including phorbol esters which activate protein kinase C induce the rapid phosphorylation of a Mr= 47 000 cytosolic protein in blood platelets (P47 or pleckstrin). This protein is well resolved by analytical 16‐BAC‐SDS two‐dimensional PAGE and was purified from platelets by preparative 16‐BAC‐SDS PAGE. Polyclonal antibodies were raised to the protein in mice and rabbits. These antisera detected a single protein with the migration of P47 on Western blots of platelet extracts, and the rabbit antisera immunoprecipitated 32P‐labelled P47 from platelet cytosol. The presence of P47 in other haematopoietic cells was determined by prelabelling them with 32P and observing increased 32P incorporation into the location of P47 on autoradiographs of 16‐BAC‐SDS analytical PAGE of cells exposed to phorbol ester. The identity of the phosphoprotein found in this location was further established by probing Western blots of SDS PAGE gels of cultured cell lines with the P47 antisera. P47 was detected in peripheral blood lymphocytes, monocytes and granulocytes (including the granulocytes of two unrelated patients with X‐linked chronic granulomatous disease). P47 was also found in HL‐60 promyelocytes (especially after differentiation with retinoic acid), U937 histiocytes, HEL leukaemia cells, and Raji ‘B’lymphoblasts. It was not detected in normal erythrocytes. K562 leukaemic cells, MOLT‐3 ‘T’lymphoblasts, or in wide range of non‐haematopoietic cell lines. We conclude that P47 is a major target for the action of phorbol ester induced phosphorylation in platelets, normal leucocytes and some haematopoietic cell lines. These cells have as their common feature the ability when stimulated to develop adhesive functions on their plasma membranes.

Prostacyclin biosynthesis in vascular endothelium is not inhibited by cyclic AMP. Studies with 3-isobutyl-1-methylxanthine and forskolin

We have previously reported (Proc. Natl. Acad. Sci. 79, 495-499, 1982) that the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), stimulates cyclic AMP accumulation and inhibits prostacyclin (PGI2) production in primary monolayer cultures of human umbilical vein endothelium. The present study was carried out to determine whether these effects are causally related. Incubation of endothelial monolayers with the diterpene, forskolin, increased the intracellular concentration of cyclic AMP by 10-fold. Despite this marked increase in cyclic AMP, neither baseline production of PGI2 nor release in response to stimulation by thrombin or the divalent cation ionophore, A23187, was affected. Both forskolin and isoproterenol were found to potentiate the effect of IBMX on cyclic AMP accumulation without causing further inhibition of PGI2 biosynthesis. Inhibition of cyclic nucleotide phosphodiesterase activity with 2,6-bis-(diethanolamino)-4-piperidinopyrimido-[5,4-d]pyrimidine increased cyclic AMP levels to the same extent as IBMX; however, this agent had no effect on PGI2 biosynthesis. These findings demonstrate that increases in the intracellular concentration of cyclic AMP have no short-term effects on PGI2 biosynthesis in vascular endothelium and suggest that inhibition of PGI2 production by IBMX is the result of some other, cyclic AMP-independent action of the drug.

Bis-4-aminoquinolines: novel triple-helix DNA intercalators and antagonists of immunostimulatory CpG-oligodeoxynucleotides

Six dimeric 2-(2-naphthyl)quinolin-4-amines with a linker between the amino groups and eight dimeric 2-(4-anilino)quinolin-4-amines linked between the anilino groups were synthesized and evaluated for their interaction with duplex/triplex DNAs and as antagonists of immunostimulatory oligodeoxynucleotides with a CpG-motif (CpG-ODN). The most powerful triple-helix DNA intercalator known to date, with high affinity toward T.A.T triplets and triplex/duplex selectivity, was found. The potent antagonism of immunostimulatory CpG-ODN by several bis-4-aminoquinolines is not related to their DNA interactions.