P-selectin inhibition therapeutically promotes thrombus resolution and prevents vein wall fibrosis better than enoxaparin and an inhibitor to von Willebrand factor.

OBJECTIVE: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT. APPROACH AND RESULTS: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed. CONCLUSIONS: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials.

Inhibition of cell adhesion by anti-P-selectin aptamer: a new potential therapeutic agent for sickle cell disease.

Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti-P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti-P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti-P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti-P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti-P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti-P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti-P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti-P-selectin aptamer may be useful as a novel therapeutic agent for SCD.

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor.

Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

ARC15105 is a potent antagonist of von Willebrand factor mediated platelet activation and adhesion.

OBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 µmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. CONCLUSIONS: The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.

Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A.

High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel-Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1-/- mice produced fewer microparticles after P-sel-Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel-Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel-Ig treatment could become a new approach to sustained control of bleeding in hemophilia.

The aptamer ARC1779 blocks von Willebrand factor-dependent platelet function in patients with thrombotic thrombocytopenic purpura ex vivo.

BACKGROUND: In thrombotic thrombocytopenic purpura (TTP), ultralarge von Willebrand factor (VWF) multimers bind platelet (PLT) glycoprotein Ib and lead to the formation of disseminated fibrin-poor, VWF-rich PLT thrombi. The aptamer ARC1779 blocks binding of the VWF A1 domain to PLT glycoprotein Ib. We evaluated whether ARC1779 inhibits the excessive VWF activity and VWF-mediated PLT function in patients with TTP. STUDY DESIGN AND METHODS: We studied the ex vivo concentration response curves for ARC1779 on PLT function analyzer (PFA-100, Dade Behring) and cone-and-plate analyzer (CPA, Impact-R) PLT function tests, agonist-induced PLT aggregation, and VWF activity of TTP patients (n = 11, three in acute phase and eight in remission) and healthy controls (n = 44). RESULTS: VWF activity and VWF-dependent PLT plug formation were increased in TTP patients relative to healthy controls, but agonist-induced PLT aggregation was not. ARC1779 blocked collagen/adenosine 5'-diphosphate (ADP)-induced PLT plug formation as measured by PFA-100 with an inhibitory concentration (IC)(100) of approximately 1 microg/mL in citrate-anticoagulated samples and approximately 3 to 4 microg/mL in hirudin-anticoagulated samples. A similar concentration of ARC1779 was necessary to block shear-dependent PLT adhesion in both TTP patients and healthy controls using the CPA assay (IC(100) of approx. 1 microg/mL for both). ARC1779 blocked VWF activity with an IC(90) of approximately 3 to 4 microg/mL in all subjects, but did not inhibit PLT aggregation by ADP, collagen, or arachidonic acid even at concentrations much greater than those that fully inhibited VWF-dependent PLT function. CONCLUSIONS: ARC1779 potently and specifically inhibits VWF activity and VWF-dependent PLT function. ARC1779 may be a promising novel therapeutic for the treatment of TTP.

Aptamers as candidate therapeutics for cardiovascular indications.

The first therapeutic aptamer was approved for human use in 2004, and a range of chemical substitutions that improve the drug-like properties of aptamers has recently been shown to increase the utility of this modality. Currently there are both anticoagulant and antithrombotic aptamers in the clinic, and additionally there are a number of earlier stage projects in which a variety of cardiovascular targets are inhibited by specific aptamers and for which a wide range of therapeutic applications has been suggested.

The aptamer ARC1779 is a potent and specific inhibitor of von Willebrand Factor mediated ex vivo platelet function in acute myocardial infarction.

ARC1779 is an aptamer, which blocks binding of the von Willebrand Factor (VWF) A1 domain to platelet GPIb receptors. VWF is increased in the elderly an in the setting of acute myocardial infarction (AMI), as reflected by increased shear-dependent platelet function. We hypothesized that ARC1779 concentration-dependently inhibits ex vivo platelet function, and that this concentration effect relationship may be shifted in patients with AMI. We studied ex vivo dose response curves for ARC1779 on VWF activity, shear-dependent platelet function, and agonist-induced platelet aggregation. We included patients with AMI on standard treatment (n = 40), young (n = 20) and elderly controls (n = 20) in this ex vivo dosing study. AMI patients displayed approximately 2-fold increased plasma levels of VWF activity as compared to controls. ARC1779 inhibited VWF activity (IC90: approximately 3-4 microg/mL) and shear dependent platelet function (Platelet Function Analyzer (PFA-100), IC50: approximately 0.5-0.9 microg/mL and Cone and Plate Analyzer (CPA), IC50: approximately 0.1-0.4 microg/mL in citrated blood) at comparable concentrations in all groups. In contrast to GPIIb/IIIa antagonists, ARC1779 did not inhibit platelet aggregation induced by ADP, collagen or arachidonic acid at concentrations (10 microg/mL) that fully inhibited VWF dependent platelet function. ARC1779 potently and specifically inhibits VWF activity and VWF dependent platelet function, even in the setting of AMI where VWF activity is increased.

First-in-human evaluation of anti von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers.

BACKGROUND: ARC1779 is a therapeutic aptamer antagonist of the A1 domain of von Willebrand Factor (vWF), the ligand for receptor glycoprotein 1b on platelets. ARC1779 is being developed as a novel antithrombotic agent for use in patients with acute coronary syndromes. METHODS AND RESULTS: This was a randomized, double-blind, placebo-controlled study in 47 healthy volunteers of doses of ARC1779 from 0.05 to 1.0 mg/kg. Pharmacodynamic effects were measured by an ELISA for free vWF A1 binding sites and by a platelet function analyzer. In terms of pharmacokinetics, the concentration-time profile of ARC1779 appeared monophasic. The observed concentration and area under the curve were dose proportional. The mean apparent elimination half-life was approximately 2 hours, and mean residence time was approximately 3 hours. The mean apparent volumes of distribution (at steady state and during terminal phase) were approximately one half the blood volume, suggesting that ARC1779 distribution is in the central compartment. The mean clearance ranged from approximately 10% to approximately 21% of the glomerular filtration rate, suggesting that renal filtration may not be a major mechanism of clearance of ARC1779. Inhibition of vWF A1 binding activity was achieved with an EC(90) value of 2.0 mug/mL (151 nmol/L) and of platelet function with an EC(90) value of 2.6 mug/mL (196 nmol/L). ARC1779 was generally well tolerated, and no bleeding was observed. Adverse events tended to be minor and not dose related. CONCLUSIONS: This is the first-in-human evaluation of a novel aptamer antagonist of vWF. ARC1779 produced dose- and concentration-dependent inhibition of vWF activity and platelet function with duration of effect suitable for the intended clinical use in acute coronary syndromes.

Characterization of the novel P-selectin inhibitor PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid] in vitro and in rodent models of vascular inflammation and thrombosis.

P-selectin plays a significant and well documented role in vascular disease by mediating leukocyte and platelet rolling and adhesion. This study characterizes the in vitro activity, pharmacokinetic properties, and the anti-inflammatory and antithrombotic efficacy of the orally active P-selectin small-molecule antagonist PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid; molecular mass, 367.83]. Biacore and cell-based assays were used to demonstrate the ability of PSI-697 to dose dependently inhibit the binding of human P-selectin to human P-selectin glycoprotein ligand-1, inhibiting 50% of binding at 50 to 125 microM. The pharmacokinetics of PSI-697 in rats were characterized by low clearance, short half-life, low volume of distribution, and moderate apparent oral bioavailability. A surgical inflammation model, using exteriorized rat cremaster venules, demonstrated that PSI-697 (50 mg/kg p.o.) significantly reduced the number of rolling leukocytes by 39% (P < 0.05) versus vehicle control. In a rat venous thrombosis model, PSI-697 (100 mg/kg p.o.) reduced thrombus weight by 18% (P < 0.05) relative to vehicle, without prolonging bleeding time. Finally, in a rat carotid injury model, PSI-697 (30 or 15 mg/kg p.o.) administered 1 h before arterial injury and once daily thereafter for 13 days resulted in dose-dependent decreases in intima/media ratios of 40.2% (P = 0.025) and 25.7% (P = 0.002) compared with vehicle controls. These data demonstrate the activity of PSI-697 in vitro and after oral administration in animal models of both arterial and venous injury and support the clinical evaluation of this novel antagonist of P-selectin in atherothrombotic and venous thrombotic indications.

Dose-dependent thrombus resolution due to oral plaminogen activator inhibitor (PAI)-1 inhibition with tiplaxtinin in a rat stenosis model of venous thrombosis.

This study aimed to evaluate a small-molecule PAI-1 inhibitor (PAI-039; tiplaxtinin) in a rodent stenosis model of venous thrombosis in a two-phase experiment. Phase 1 determined the efficacy of tiplaxtinin against Lovenox (LOV), while phase 2 determined the dose-dependent efficacy. For both phases, drug treatment began 24 hours after surgically induced venous thrombosis and continued for four days. Phase 1 animals (n = 24) receiving low-dose (LD; 1 mg/kg oral gavage) PAI-1 inhibitor demonstrated a 52% decrease in thrombus weight (TW) versus controls (p < 0.05) with significant reductions in active plasma PAI-1, while the high-dose (HD; 10 mg/kg oral gavage) group demonstrated a 23% reduction in TW versus controls. Animals treated subcutaneously with LOV (3 mg/kg) showed a 39% decrease in TW versus controls (p < 0.05). Coagulation tests (aPTT and TCT) were significantly different in LOV compared to PAI-1 inhibitor groups. PAI-039 treatment was also associated with significantly increased return of inferior vena cava blood flow four days post-thrombosis versus controls (p < 0.05). In phase 2 (n = 30), TW was reduced from the 0.5 mg/kg to 5 mg/kg experimental groups, with the 10 mg/kg group demonstrating a paradoxical increase. The 5 mg/kg group showed statistically significant decreases in TW versus controls after four treatment days (p < 0.05). This is the first study to demonstrate dose related effects of PAI-039 on increasing thrombus resolution and inferior vena cava blood flow without adverse effects on anti-coagulation in a rat stenosis model of venous thrombosis.

Prophylactic P-selectin inhibition with PSI-421 promotes resolution of venous thrombosis without anticoagulation.

P-selectin inhibition has been evaluated as a therapeutic for prevention and treatment of venous thrombosis. In this study, a novel oral small-molecule inhibitor of P-selectin, PSI-421, was evaluated in a baboon model of stasis induced deep vein thrombosis (DVT). Experimental groups included i) primates receiving a single oral dose of 1 mg/kg PSI-421 two days prior and continued six days after thrombosis (n = 3); ii) primates receiving a single daily subcutaneous dose of 0.57 mg/kg enoxaparin sodium two days prior and continued six days post thrombosis (n = 3); and iii) primates receiving no treatment (n = 3). PSI-421 treated primates had greater percent vein reopening and less vein wall inflammation than the enoxaparin and controls at day 6. Microparticle tissue factor activity (MPTFA) was significantly lower in the animals receiving PSI-421 immediately after thrombosis (T+6 hours day 0) suggesting lower potential for thrombogenesis in these animals. PSI-421 also reduced soluble P-selectin levels versus controls at T+6 hours day 0, day 2 and 6. Experimental animals in any group showed no adverse effects on coagulation. This study is the first to demonstrate a reduction in MPTFA associated with vein reopening and reduced vein inflammation due to oral P-selectin inhibition in a baboon model of DVT.

2-(4-Chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[H]quinoline-4-carboxylic acid (PSI-697): identification of a clinical candidate from the quinoline salicylic acid series of P-selectin antagonists.

P-selectin-PSGL-1 interaction causes rolling of leukocytes on the endothelial cell surface, which subsequently leads to firm adherence and leukocyte transmigration through the vessel wall into the surrounding tissues. P-selectin is upregulated on the surface of both platelets and endothelium in a variety of atherosclerosis-associated conditions. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of atherosclerosis. High-throughput screening and subsequent analoging had led to the identification of compound 1 as the lead candidate. Herein, we report the continuation of this work and the discovery of a second-generation series, the tetrahydrobenzoquinoline salicylic acids. These compounds have improved pharmacokinetic properties, and a number of them have shown oral efficacy in mouse and rat models of atherogenesis and vascular injury. The lead 31 (PSI-697), is currently in clinical development for the treatment of atherothrombotic vascular events.

The von Willebrand factor antagonist (GPG-290) prevents coronary thrombosis without prolongation of bleeding time.

The interaction between von Willebrand factor (VWF) and platelet glycoprotein Ibalpha (GPIbalpha) is a critical step that allows platelet adhesion, activation and subsequent thrombus formation to the injured vessel wall under high-shear conditions. In this study, we sought to investigate 1) whether GPG-290, a recombinant human GPIbalpha chimeric protein, would prevent thrombosis in a canine model of coronary thrombosis by blocking VWF-GPIbalpha interaction; and 2) whether desmopressin (DDAVP), a VWF release stimulant, could reduce the prolonged bleeding time caused by a 10x efficacious dose of GPG-290. The antithrombotic efficacy of GPG-290 was evaluated by the in-vivo ability to prevent cyclic flow reductions (CFRs) and ex-vivo inhibition of platelet adhesion/aggregation reflected by prolongation of Platelet Function Analyzer (PFA-100) collagen/ADP closure time. The anti-hemostatic effect was assessed by template bleeding time. GPG-290 at doses of 25, 50 and 100 microg/kg abolished CFRs in 67%, 100% and 100% of the treated dogs without bleeding time prolongation, respectively; GPG-290 dose-dependently prolonged the ex-vivo collagen/ADP-closure time, while it had no effects on plasma VWF antigen level (VWF:Ag) and VWF-collagen binding activity (VWF:CB); the prolonged template bleeding time caused by 500 microg/kg of GPG-290 was prevented by intravenous infusion of DDAVP (0.3 microg/kg). In conclusion, GPG-290 appears to be an effective agent for treating arterial thrombosis without bleeding time prolongation.

Resolution of venous thrombosis using a novel oral small-molecule inhibitor of P-selectin (PSI-697) without anticoagulation.

P-selectin inhibition has been shown to decrease thrombogenesis in multiple animal species. In this study, we show that a novel oral small-molecule inhibitor of P-selectin, PSI-697, promotes thrombus resolution and decreases inflammation in a baboon model of venous thrombosis. Experimental groups consisted of the following: 1) primates receiving a single oral dose of PSI-697 (30 mg/kg) daily starting three days pre-iliac vein balloon occlusion, and continued for six days; 2) primates receiving a single treatment dose of a low-molecular-weight-heparin (LMWH) (1.5 mg/kg) daily starting one day pre-iliac balloon occlusion, and continued for six days; and 3) primates receiving a single oral dose of a vehicle control daily starting three days pre-iliac vein balloon occlusion, and continued for six days. Animals receiving PSI-697, although thrombosed after balloon deflation, demonstrated greater than 80% vein lumen opening over time, with no opening (0%) for vehicle control (p < 0.01). LMWH opening evident after balloon deflation slightly deteriorated over time compared to PSI-697. PSI-697 therapy also significantly decreased vein wall inflammation determined by magnetic resonance venography (MRV). Importantly, this beneficial opening occurred without measured anticoagulation. Animals receiving PSI-697 demonstrated significantly increased plasma D-dimer levels versus LMWH and control animals six hours post thrombus induction (p < 0.01). This study is the first to demonstrate the effectiveness of oral P-selectin inhibition to modify venous thrombogenesis, increase vein lumen opening, and decrease inflammation in a large animal model.

Pharmacologic inhibition of platelet vWF-GPIb alpha interaction prevents coronary artery thrombosis.

Under high shear arterial blood flow von Willebrand Factor (vWF) binds the platelet receptor glycoprotein (GP) Ibalpha, leading to platelet adhesion, activation and thrombosis. Blockade of vWF-GPIb alpha interactions by GPG-290 was investigated in a canine model of coronary artery thrombosis alone and in combination with clopidogrel. GPG-290 (100 microg/kg, n=6; 500 microg/kg, n=6) prolonged time to thrombotic occlusion (TTO) to 105+/-34 and 156+/-23 (p<0.05) min, respectively compared to the saline treated control group (32+/-6 min, n=6). Patency of the injured vessel was sustained in 1/6 (100 microg/kg) and 3/6 vessels (500 microg/kg) 4 hours after injury, in contrast to 0/6 in the control group. There was an increase in bleeding after the 500 microg/kg dose, but only at the 1 hr time point. Clopidogrel was studied in two dosing regimens representing either a clinical pretreatment regimen (PTR) of 4.3 mg/kg on day -2 followed by 1.1 mg/kg daily for 2 days prior to the procedure or pre-procedural loading dose regimen (LDR) of 4.3 mg/kg 3 hr pre-procedure. The PTR and LDR clopidogrel treatments prolonged TTO to 98.2+/-30.0 min and 136.1+/-39.5 min (p<0.05), and sustained patency in 1/6 and 4/8 vessels, respectively. However, template bleeding time in the LDR clopidogrel group was sustained higher than the control group. The combination of PTR clopidogrel and GPG-290 (100 microg/kg) prolonged TTO equivalent to LDR clopidogrel alone (141.4 +/- 35.1 min) and sustained patency in 3/7 dogs, without increased bleeding while LDR clopidogrel combined with 100 microg/kg GPG-290 prevented occlusion in 5/8 dogs and further prolonged TTO (173.5+/-32.6 min) but was associated with increased bleeding compared to control. GPG-290 is an antithrombotic agent that may be combined with lower doses of clopidogrel to yield similar antithrombotic efficacy as higher loading doses.

Prevention of in-stent restenosis via reduction of thrombo-inflammatory reactions with recombinant P-selectin glycoprotein ligand-1.

The binding of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) to platelet P-selectin is central to post-angioplasty restenosis. Although intracoronary stents limit the mechanical component of restenosis, they cause marked thrombo-inflammation and neointimal proliferation leading to greater late luminal loss. We sought to demonstrate that P-selectin antagonism, using recombinant PSGL-1 (rPSGL-Ig), is effective in reducing platelet-leukocyte reactions and in-stent restenosis in double-injured porcine coronary arteries. Two weeks after initial injury by angioplasty to the coronary arteries, stents were implanted at the injury-induced lesion site, 15 min after an i.v. bolus administration of a vehicle or rPSGL-Ig (1 mg/kg). Four weeks later, adhesion of (51)Cr-platelets and (111)In-neutrophils and histomorphometric analyses were performed. In-stent residual lumen was almost 3 fold larger in rPSGL-Ig-treated arteries (3.1 +/- 0.4 mm(2)) as compared to control (1.1 +/- 0.2 mm(2)), which correspond to 64% vascular stenosis in control with no change in rPSGL-Ig animals. For a similar injury score, in-stent neointima was significantly reduced by 30 to 40% in the rPSGL-Ig group and quantitative coronary angiography showed a significant 35% reduction in late lumen loss. These effects of rPSGL-Ig were associated with a respective 70% and 53% reduction in platelet and neutrophil adhesion. In conclusion, pretreatment with rPSGL-Ig reduces thrombo-inflammatory responses, neointimal proliferation, and in-stent restenosis. P-selectin antagonism offers a promising therapy to improve clinical outcomes of coronary stenting.

Recombinant soluble P-selectin glycoprotein ligand-Ig (rPSGL-Ig) attenuates infarct size and myeloperoxidase activity in a canine model of ischemia-reperfusion.

The role of P-selectin in the process of reperfusion injury was evaluated using a recombinant soluble P-selectin glycoprotein ligand-Ig (rPSGL-Ig) in a canine coronary artery balloon occlusion model. rPSGL-Ig (1 mg/kg) or saline was given as an intravenous bolus 15 min before balloon deflation. Balloon occlusion time was 90 min followed by either 120 min or 7 days reperfusion. Infarct size was significantly reduced in the treatment group when expressed either as percentage of the area at risk or as absolute infarct size. Histological analysis showed that extensive myocardial injury and neutrophil infiltration were reduced by rPSGL-Ig. Myeloperoxidase activity (MPO) was significantly reduced in the risk area in the rPSGL-Ig group. Left ventricular ejection fraction was significantly less impaired during the first 24 h after reperfusion in the rPSGL-Ig group, although there was no difference by 7-day follow-up. Thus, administration of rPSGL-Ig decreases myocardial injury and inflammatory response for at least 7 days after reperfusion of ischemic myocardium.

Drug-drug interaction of the anti-TFPI aptamer BAX499 and factor VIII: studies of spatial dynamics of fibrin clot formation in hemophilia A.

BACKGROUND: In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. OBJECTIVE: To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro. METHODS: Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6pmole/m(2) by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C<5%, but the effect was only 25% if FVIII:C>30%). CONCLUSIONS: The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.

Recent advances in the development of coagulation factors and procoagulants for the treatment of hemophilia.

Hemophilia is a family of rare bleeding disorders. The two primary types, hemophilia A and hemophilia B, are caused by recessive X-chromosome linked mutations that result in deficiency of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Clinically, hemophilia is manifested by spontaneous bleeding, particularly into the joints (haemarthrosis) and soft tissue, and excessive bleeding following trauma or surgery. The total overall number of hemophilia patients worldwide is approximately 400,000, however only about 100,000 of these individuals are treated. The first treatment of hemophilia was initiated when it was determined that the clotting deficiency could be corrected by a plasma fraction taken from normal blood. The discovery of factor VIII enrichment by cryoprecipitation of plasma opened a new era of therapy which eventually led to the production of factor concentrates and the subsequent development of highly purified forms of plasma factors. The most significant improvements have been the availability of recombinant forms of factors VIII and IX. Unfortunately, recombinant factors still retain some of the limitations of plasma concentrates. These limitations include development of antibody responses in patients and the relatively short half-life of the molecules requiring frequent injection to maintain effective concentration. Treatment beyond replacement of native factors has been tried. They include the development of modified factor VIII and IX molecules with improved potency, stability and circulating half-life and enhancement of a prothrombotic responses and/or stabilization of coagulation factors via inhibition of key negative regulatory pathways. These approaches will be reviewed in this commentary.