Inhibited Platelet Deposition Research Articles

Activation of Most Toll-Like Receptors in Whole Human Blood Attenuates Platelet Deposition on Collagen under Flow.

Platelets have toll-like receptors (TLRs); however, their function in thrombosis or hemostasis under flow conditions is not fully known. Thrombin-inhibited anticoagulated whole blood was treated with various TLR agonists and then perfused over fibrillar collagen using microfluidic assay at venous wall shear rate (100 s-1). Platelet deposition was imaged with fluorescent anti-CD61. For perfusion of whole blood without TLR agonist addition, platelets rapidly accumulated on collagen and eventually occluded the microchannels. Interestingly, most of the tested TLR agonists (Pam3CKS4, MALP-2, polyinosinic-polycytidylic acid HMW, imiquimod, and CpG oligodeoxynucleotides) strongly reduced platelet deposition on collagen, while only the TLR4 agonist endotoxin lipopolysaccharide (LPS) enhanced deposition. Following 90 sec of deposition under flow of untreated blood, the addition of various TLR-7 agonists (imiquimod, vesatolimod, and GSK2245035) all caused immediate blockade of further platelet deposition. Since TLR signaling can activate nuclear factor-kappaB (NF-κB), the IKK-inhibitor (IKK inhibitor VII) and NF-κB inhibitor (Bay 11-7082) were tested. The IKK/NF-κB inhibitors strongly inhibited platelet deposition under flow. Furthermore, addition of Pam3CSK4 (TLR1/2 ligand), MALP-2 (TLR2/6 ligand), and Imquimod (TLR7 ligand) reduced phosphotidylserine (PS) exposure. Activation of TLR1/2, TLR2/6, TLR3, TLR7, and TLR9 in whole blood reduced platelet deposition under flow on collagen; however, LPS (major Gram negative bacterial pathogenic component) activation of LTR4 was clearly prothrombotic.

Anti-GPVI Fab reveals distinct roles for GPVI signaling in the first platelet layer and subsequent layers during microfluidic clotting on collagen with or without tissue factor

The collagen receptor glycoprotein VI (GPVI) drives strong platelet activation, however its role at later stages of clotting remains less clear. Controlled timing of addition of anti-human GPVI Fab (clone E12) with microfluidic venous whole blood flow over collagen (± lipidated tissue factor, TF) produced distinct effects on platelets, fibrin, P-selectin exposure, and phosphatidylserine (PS) exposure. On collagen alone, Fab present initially potently reduced platelet deposition on collagen, while Fab added 90 s after initial platelet deposition, stopped subsequent platelet accumulation (despite the absence of fibrin). With thrombin generation via TF, Fab added at either t = 0 or 90 s had no effect on platelet deposition. However, Fab added initially, but not at 90-s, blocked fibrin formation. Gly-Pro-Arg-Pro ablated fibrin formation without effect on platelet accumulation (regardless of Fab added at t = 0 or 90 s), indicating thrombin signaling can suffice over GPVI signaling. Still, Fab moderately reduced P-selectin exposure with thrombin present and fibrin absent. On collagen/TF, Fab present initially ablated PS exposure, but had no effect when added 30 to 90-s later. The thrombin generated via PS exposure had an important role in driving platelet deposition in the presence of Fab, since inhibition of PS via annexin V binding in the presence of Fab significantly inhibited platelet deposition. We conclude GPVI signaling in the first platelet layer on collagen dictates thrombin and fibrin production, but the role of GPVI at subsequent times after formation of the first monolayer is obscured by thrombin-induced signaling.

Ibrutinib Inhibits BMX-Dependent Endothelial VCAM-1 Expression In Vitro and Pro-Atherosclerotic Endothelial Activation and Platelet Adhesion In Vivo.

Inflammatoryactivation of the vascular endothelium leads to overexpression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), contributing to the pro-thromboticstate underpinning atherogenesis. While the role of TEC family kinases (TFKs) in mediating inflammatory cell and platelet activation is well defined, the role of TFKs in vascular endothelial activation remains unclear. We investigated the role of TFKs in endothelial cell activation in vitro and in a nonhuman primate model of diet-induced atherosclerosis in vivo. In vitro, we found that ibrutinib blocked activation of the TFK member, BMX, by vascular endothelial growth factors (VEGF)-A in human aortic endothelial cells (HAECs). Blockade of BMX activation with ibrutinib or pharmacologically distinct BMX inhibitors eliminated the ability of VEGF-A to stimulate VCAM-1 expression in HAECs. We validated that treatment with ibrutinib inhibited TFK-mediated platelet activation and aggregation in both human and primate samples as measured using flow cytometry and light transmission aggregometry. We utilized contrast-enhancedultrasound molecular imaging to measure platelet GPIbα and endothelialVCAM-1 expression in atherosclerosis-prone carotid arteries of obese nonhuman primates. We observed that the TFK inhibitor, ibrutinib, inhibited platelet deposition and endothelial cell activation in vivo. Herein we found that VEGF-A signals through BMX to induce VCAM-1 expression in endothelial cells, and that VCAM-1 expression is sensitive to ibrutinib in vitro and in atherosclerosis-prone carotid arteries in vivo. These findings suggest that TFKs may contribute to the pathogenesis of atherosclerosis and could represent a novel therapeutic target.

Development of Coagulation Factor XII Antibodies for Inhibiting Vascular Device-Related Thrombosis.

Vascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis. Generate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity. Monoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation. A FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate. Selective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.

Covalently‐Attached, Surface‐Eroding Polymer Coatings on Magnesium Alloys for Corrosion Control and Temporally Varying Support of Cell Adhesion

AbstractFor degradable magnesium (Mg) alloy‐based stents it would be desirable to delay early corrosion to maintain mechanical strength. Similarly, early after stent placement reduced thrombogenicity is an important feature, while chronically, endothelial cell adhesion and vessel integration are desirable. In this study, surface eroding polymers of amino‐grafted poly(1,3‐trimethylene carbonate) (PTMC‐NH2) and PTMC‐NH2 combined with sulfobetaine bearing polymer PSB (PTMC‐NHCO‐PSB) are developed, and these polymers are covalently attached onto 6‐phosphonohexanoic acid (PHA)‐coated AZ31 Mg alloy surfaces in sequence. In vitro degradation testing in ovine plasma shows PTMC, PTMC‐NH2, and PTMC‐NHCO‐PSB cast films experience a gradual thickness and mass loss with maintenance of smooth surfaces, confirming surface erosion behavior. The PTMC‐NH2 polymer is firmly bound to the PHA‐modified AZ31 surface and demonstrates a resistance to peeling. PTMC, PTMC‐NH2, and PTMC‐NHCO‐PSB coated AZ31 have a lower corrosion rate versus polylactide‐co‐glycolide coated and untreated AZ31. PTMC‐NHCO‐PSB coated AZ31 inhibits platelet deposition and smooth muscle cell adhesion and growth, but after 2‐week immersion in plasma, this surface supports endothelial cell adhesion and growth. These results suggest PTMC‐NHCO‐PSB surface eroding coating offers a means of controlling corrosion while providing a temporally varying bio‐functionality for biodegradable vascular stent applications.

Src family kinases inhibition by dasatinib blocks initial and subsequent platelet deposition on collagen under flow, but lacks efficacy with thrombin generation

Kinase inhibitors can pose bleeding risks as platelet signaling evolves during clotting. Using microfluidics (200 s−1 wall shear rate) to perfuse Factor XIIa-inhibited or thrombin-inhibited whole blood (WB) over collagen ± tissue factor (TF), we explored the potency of the Src family kinase (SFK) inhibitor dasatinib or the spleen tyrosine kinase (Syk) inhibitor GS-9973 present at clot initiation or added after 90 s (via rapid switch to inhibitor-pretreated WB). When initially present, dasatinib potently inhibited platelet deposition on collagen (no TF). Furthermore, dasatinib immediately inhibited subsequent platelet deposition when introduced 90 s after clot initiation. However, when thrombin was generated, dasatinib was markedly less potent against platelet deposition on collagen/TF (but blocked fibrin deposition) and had no effect when added 90 s after clot initiation. Similarly, dasatinib added at 90 s had no effect on clotting on collagen/TF when fibrin was also blocked with Gly-Pro-Arg-Pro, indicating that strong thrombin-induced signaling (but not fibrin-induced signaling) can bypass the SFK inhibition at later times. The Syk inhibitor GS-9973 was less potent than dasatinib when present initially, but inhibited clot growth when added at 90 s, even in the presence of thrombin (±fibrin). Interestingly, the active form (R-406) of fostamatinib inhibits platelet function in only 2 0f 5 healthy blood samples. SFK-inhibitors may have reduced antithrombotic activity and reduced bleeding risks in settings of high TF and local thrombin generation. For oncology patients, SFK-inhibitors like dasatinib may have reduced antithrombotic activity and reduced bleeding risk in settings of local thrombin generation.

Mast cell-derived heparin proteoglycans as a model for a local antithrombotic.

Mast cell-derived heparin proteoglycans (HEP-PG) reside in vascular tissue and serve as a local antithrombotic. Heparin, as used clinically, is isolated from its protein backbone from porcine and bovine gut mucosa. The isolated heparin is an anticoagulant; however, when bound to a protein carrier the heparin conjugates will become antiplatelet agents by inhibiting collagen-induced platelet aggregation and procoagulant activity, as distinct from soluble heparin. HEP-PG, whether soluble or immobilized to a surface, inhibit platelet deposition on the collagen surface in flowing blood. Mimics of HEP-PG, can be tailored to molecules carrying both antiplatelet and anticoagulant (APAC) properties. These molecules can target the vascular injury site and take residence there. Inhibition of thrombus growth using these APACs under these conditions has been demonstrated in several animal models. Although efficacious for antiplatelet and anticoagulant effects, the bleeding time is shorter with APACs than with unfractionated heparin, suggestive of beneficial efficacy/safety ratio. These strategies may be utilized in drug development, where many vascular injury-related problems can be tackled locally.

Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (∼10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. 19F MRI confirmed colocalization of particles with arterial thrombi, and quantitative 19F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes.

Rapid on-chip recalcification and drug dosing of citrated whole blood using microfluidic buffer sheath flow.

Millions of clotting tests each year require recalcification of blood treated with sodium citrate, a calcium chelator that prevents prothrombinase assembly. We validated a converging trifurcated microfluidic device to measure platelet and fibrin accumulation following on-chip recalcification of citrated whole blood. Recalcification was accomplished by sheathing the blood with Ca2+ buffer. Fluorescein rapidly diffused across the buffer-blood interface (achieving 62.5% of maximum centerline concentration within ~4 cm of flow), while albumin remained relatively unchanged in blood due to its lower diffusivity (<20% decrease). Since Ca2+ diffuses faster than fluorescein, full recalcification of whole blood was achieved within ~1 cm of flow prior to encountering a collagen/tissue surface. Platelet and fibrin were reduced by 87.3% and 99.0%, respectively, when the sheath buffer was Ca2+-free. A 30-min preincubation of citrated whole blood prior to on-chip recalcification increased platelet (159%) and fibrin (86.6%) deposition, compared to 5-min preincubation, likely due to factor XIIa generation in citrated blood. The P2Y1 inhibitor, MRS-2179, was delivered by diffusion into flowing blood and inhibited platelet deposition on collagen with a calculated IC50 of 0.155 μM. On-chip recalcification and drug dosing of citrated blood allows for assays of platelet function in a whole blood milieu under flow.

Antithrombotic activity of protein S infused without activated protein C in a baboon thrombosis model

Protein S (ProS) is an essential plasma protein that enhances the anticoagulant activity of activated protein C (APC). In vitro , purified native human Zn2+-containing ProS also exerts direct anticoagulant activity by inhibiting prothrombinase and extrinsic FXase activities independently of APC. We investigated antithrombotic effects of ProS infused without APC in a baboon shunt model of thrombogenesis that employs a device consisting of arterial and venous shear flow segments. In in vitro experiments, the Zn2+-containing human ProS used for the studies displayed >10-fold higher prothrombinase inhibitory activity and anticoagulant activity in tissue factor-stimulated plasma, and four-fold higher inhibition of the intrinsic pathway than the Zn2+-deficient ProS used. In the thrombosis model, ProS (33 μg/minute for 1 hour) or saline was infused locally; platelet and fibrin deposition in the shunt were measured over 2 hours. During experiments performed at 50 ml/minute blood flow, Zn2+-containing ProS inhibited platelet deposition 73-96% in arterial-type flow segments and 90-99% in venous-type flow segments; Zn2+-deficient ProS inhibited platelet deposition 52% in arterial-type flow segments and 65-73% in venous-type flow segments. At 100 ml/min blood flow rate, Zn2+-containing ProS inhibited platelet deposition by 39% and 73% in the respective segments; Zn2+-deficient ProS inhibited platelet deposition by 5% and 0% in the respective segments. Zn2+-containing ProS suppressed fibrin deposition by 67-90%. Systemic APC-independent ProS activity was significantly increased and thrombin-antithrombin complex levels were significantly decreased after infusion of ProS. Thus, infused human Zn2+-containing ProS is antithrombotic in primates, and may have therapeutic potential even in protein C-deficient human patients.

Salvianolic Acid B inhibits platelet adhesion under conditions of flow by a mechanism involving the collagen receptor α2β1

Salvianolic acid B (SAB) is a component of Danshen, a herb widely used in Chinese medicine, and was previously shown to exert a number of biological activities including inhibition of platelet function, but the exact mechanisms involved are unclear. SAB dose-dependently inhibited platelet deposition from flowing, anticoagulated whole blood to immobilized collagen at both venous and arterial shear rate, whereas platelet deposition to immobilized fibrinogen was not affected. The inhibitory effect of SAB on platelet adhesion to collagen was independent of αIIbβ3, since SAB still inhibited platelet deposition in the presence of a αIIbβ3-blocking peptide. SAB inhibited static platelet adhesion to a synthetic peptide specific for the collagen receptor α2β1, whereas platelet adhesion to a glycoprotein VI-specific peptide was not affected. SAB inhibited binding of an antibody against α2β1 to platelets as studied by flow cytometry, and inhibited the interaction of soluble α2β1 to immobilized collagen in a solid phase binding assay. These combined results indicate that SAB inhibits platelet adhesion to immobilized collagen by interfering with the collagen receptor α2β1.

Comparison of PD0348292, a selective factor Xa inhibitor, to antiplatelet agents for the inhibition of arterial thrombosis

The objective of this study was to determine if orally-administered PD0348292, a direct specific factor Xa inhibitor, inhibits thrombosis following porcine carotid arterial injury comparably to aspirin or clopidogrel alone or in combination. We further sought to determine whether the antithrombotic efficacy in vivo could be predicted using an ex-vivo perfusion chamber. Oral treatments included: PD0348292 (0.4, 0.9, or 4.3 mg/kg); PD0348292 (0.4 mg/kg) plus aspirin (325 mg); aspirin; clopidogrel (75 mg); aspirin plus clopidogrel; or vehicle (n = 6-10/group). Aspirin and clopidogrel were administered 27 and four hours pre-injury and PD0348292 or vehicle was administered four hours pre-injury. Both carotid arteries were crush-injured, and thrombus was measured by detection of (111)In-platelets over 30 minutes. Prior to injury, the antithrombotic efficacy was assessed by ex-vivo perfusion chamber platelet deposition. PD0348292 produced dose-dependent prothrombin time (0.9- to 2.9-fold) and aPTT (1.4- to 2.5-fold) prolongations. Bleeding times were significantly prolonged in each active drug group compared to vehicle, but were not significantly different between drug groups. PD0348292 significantly inhibited arterial platelet deposition (x10(6)/cm(2)) at 4.3(549 +/- 1,066), 0.9 (399 +/- 162) and 0.4 mg/kg (531 +/- 470) compared to vehicle (2,242 +/- 1,443). Aspirin (992 +/- 973), clopidogrel (537 +/- 483), clopidogrel plus aspirin (228 +/- 66) or PD0348292 plus aspirin (558 +/- 317) also significantly inhibited platelet deposition, although these values were not significantly different than with any dose of PD348292. Perfusion chamber platelet deposition correlated significantly with in-vivo anti-thrombotic response. In conclusion, PD0348292 inhibited arterial thrombosis comparable to aspirin plus clopidogrel. Perfusion chamber methodology may be useful in predicting in-vivo antithrombotic efficacy.

Antithrombotic Activity of PRT54021, a Potent Oral Direct Factor Xa Inhibitor, Can Be Monitored Using a Novel Prothrombinase Inhibition Bioassay.

Coagulation factor Xa (fXa) is a validated target for antithrombotic therapy and there are several on-going clinical trials testing direct fXa inhibitors. Current measures for monitoring coagulation status [activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), activated clotting time (ACT), anti fXa units] have been developed for existing anticoagulants (i.e., heparins and warfarin). The available tests are not sensitive enough to evaluate therapeutic concentrations of direct fXa inhibitors. Since, the true target of fXa inhibitors is the membrane associated prothrombinase complex, we hypothesized that an assay measuring thrombin generation would be a superior measure of the level of anti-coagulation achieved in patients dosed with direct fXa inhibitors. PRT54021 (PRT021), an orally bioavailable fXa inhibitor in advanced stages of clinical development (Phase II), was used to validate this hypothesis. PRT021 is an active site directed, competitive inhibitor of human fXa (Ki=117pM ) and exhibits a >86,000 fold specificity against related proteases such as thrombin, factor VIIa, factor IXa, activated protein C, tissue plasminogen activator, plasmin and trypsin. PRT021 is a potent inhibitor of the purified prothrombinase complex (Ki=801pM). Evaluation in a whole blood prothrombinase assay was carried out to compare PRT021 to fondaparinux, an indirect inhibitor of fXa. Like fondaparinux, PRT021 dose-dependently inhibited platelet mediated prothrombinase activity in this test system. To measure prothrombinase inhibition, we developed a new bioassay which used a fluorogenic thrombin substrate to quantitate the amount of thrombin produced upon addition of tissue factor to human plasma. In order to determine if this new bioassay predicts in vivo antithrombotic activity, we tested PRT021 in a baboon model of arteriovenous shunt thrombosis. 111In labeled platelet and 125I fibrinogen deposition on the thrombogenic device were used as indicators of thrombotic activity. Dose-dependent inhibition of venous thrombosis was observed at four doses (0.05, 0.12, 0.21 and 0.49mg/kg) of PRT021. Ex vivo measurements of plasma thrombin generation, aPTT, PT, ACT, and anti fXa units were performed during the time course. In contrast to the observed antithrombotic activity (30 to 90% inhibition of platelet deposition, 0 to 87% inhibition of fibrin deposition), there were minimal extension of clotting parameters upon PRT021 treatment. Anti fXa units were below the limit of quantitation for the three lower doses and 0.31 Units/ml at the highest dose. Template bleeding times were not perturbed for any of the PRT021 treated animals. The only ex vivo parameter that correlated to the antithrombotic activity was the dose proportional inhibition (correlation coefficient R2=0.99) of plasma thrombin generation; which ranged from 13% inhibition at the lowest dose to 72% at the highest dose. Thus we have established that this new prothrombinase bioassay predicts in vivo antithrombotic activity. PRT021 is currently in clinical development for prevention and treatment of venous thromboembolic diseases. On-going work with plasma from PRT021 treated patients will verify if the bioassay correlates with clinical endpoints.

Protein S Is Antithrombotic In Vivo Independent of APC.

Protein S in plasma has APC-independent, direct anticoagulant activity. Preparations of purified protein S vary in this activity, depending on purification methods. Plasma and purified protein S form multimers that do not appear to differ in ability to inhibit prothrombinase activity (Heeb et al J Thromb Haemost 2006) except in the case of protein S purified using quarternary amine resins. We sought to determine if protein S purified by different methods has direct antithrombotic activity in vivo using a well-characterized baboon thrombosis model. A silicon rubber graft was placed between the femoral vein and artery and included a thrombogenic dacron segment that simulated thrombus formed under arterial flow conditions, followed by a teflon chamber of larger diameter that simulated thrombus formed under venous flow, then a region in which a propagating tail thrombus formed. 111In-labeled autologous platelets and 125I-labeled fibrinogen were infused prior to opening the graft. Protein S (2 mg/hr) or saline was infused 1 cm upstream circumferentially along the wall of the shunt for 1 hr after opening the graft. Deposition of platelets and fibrin was monitored for 2 hr. Eleven experiments were performed, using four different baboons. During the 1 hr infusion, affinity-purified recombinant and plasma-derived protein S inhibited platelet deposition by up to 62% in the arterial segment, up to 89% in the venous segment, and up to 100% in the propagating tail thrombus. Recombinant protein S purified by a different method using quaternary resins had weaker activity, consistent with in vitro clotting assay data. Protein S was more effective in experiments performed at flow rates of 40 ml/hr than in experiments performed at flow rates of 100 ml/hr. In other experiments, plasma-derived protein S was infused in the absence and presence of systemically administered antibody that blocked protein C activation (HPC4). Ex vivo blood samples showed that the ability of protein C to be activated was blocked by 91% or more by HPC4. During the 1 hr infusion, protein S suppressed at least 95% of platelet deposition in the absence of HPC4. In presence of HPC4, protein S suppressed platelet deposition by 79% in the dacron segment, and by 97% in the other segments. After protein S infusion was discontinued, a prothrombotic effect due to HPC4 was observed, in that platelet deposition occurred in the graft somewhat faster than in the saline control without protein S and HPC4, and the shunt occluded 20 min after cessation of protein S. Final fibrin deposition 2 hr after the beginning of the experiments was suppressed by protein S by 34 and 67% in the dacron segments and by 83 and 90% in the chambers, in the presence and absence of HPC4, respectively. Plasma from blood taken downstream of the shunt had modest but significant elevation of direct anticoagulant protein S activity, measured in endogenous thrombin potential assays, after local protein S infusion in the presence and absence of HPC4. APC cofactor activity was not significantly changed in any samples, but there was a trend to higher values after local protein S infusion. These experiments are the first to demonstrate that purified protein S has APC-independent, direct antithrombotic activity in vivo in primates, and suggest therapeutic potential for protein S as an antithrombotic agent.

Comparison of Enoxaparin to the Thrombin Variant Protein C Activator, W215A/E217A, in a Primate Model of Thrombosis.

Antithrombotic therapy by thrombomodulin-dependent activation of endogenous protein C might be safer than currently used therapies, since endogenous activated protein C (APC) is generated and probably concentrated on the endothelial surface and not in blood that escapes the blood vessels during injury. We compared the antithrombotic and antihemostatic activities of a protein C selective recombinant double mutant human thrombin (W215A/E217A; WE) that activates endogenous protein C on the endothelial surface, in vivo, with a low molecular weight heparin, enoxaparin in awake non anticoagulated baboons. Thrombosis was initiated by interposing a two part device consisting of a 2cm, 4mm ID, polyethylene terephthalate vascular graft (DVG) followed by a 2cm silicone tubing chamber, 9mm ID, into a chronic femoral arteriovenous shunt in baboons. Upon initiation of blood flow at 100mL/min, wall shear rate of 265sec−1 in the 4mm ID segment (arterial type flow) and 29sec−1 in the expansion chamber (venous type flow), platelet thrombus growth was monitored by gamma camera imaging of autologous 111In-labeled platelets for 1 hour. Fibrin accumulation was quantified by homologous 125I-labeled fibrinogen. Bleeding time and aPTT were monitored. WE was administered as a loading IV bolus (1/3 dose) 10 minutes before thrombus initiation followed by maintenance infusion (2/3 dose) for the duration of the study at 3 dose levels: 1.8 μg/kg (dose-1), 3.75 μg/kg (dose-2) and 7.5 μg/kg (dose-3). Results were compared to controls given normal saline and to 3 doses of IV enoxaparin (1/2 dose loading + 1/2 dose maintenance): 0.3 mg/kg (dose-1), 0.6 mg/kg (dose-2) and 1.2mg/kg (dose-3) (3–5 animals in each study group). WE inhibition of platelet deposition in the chamber was dose dependent and reduced by 57%, 76% and 95% for dose 1, 2 and 3 respectively, compared to controls (p < 0.05 each). Enoxaparin likewise reduced thrombosis in the chamber by 49%, 83% and 91% (p< 0.05 each) respectively at the 3 doses tested. Fibrin accumulation paralleled the platelet deposition data in the chamber. Thrombus growth on DVG was only reduced at dose level 3 for both WE and enoxaparin by 18% and 47%, respectively as determined by platelet deposition. Fibrin accumulation was reduced by 35% by WE and 51% for enoxaparin at dose 3 on the arterial thrombosis segment. Bleeding times did not increase significantly with any of the treatments except for dose-3 of enoxaparin which prolonged the bleeding time from 3.5±1.3 minutes to 5.3 ± 1.4 minutes. Prolongation of APTT was 1.14, 1.33, and 2.04 fold for WE doses 1, 2 and 3, respectively and enoxaparin prolonged the APTT by 1.25, 1.6 and 1.9 fold at dose 1, 2 and 3, respectively. We conclude that low dose WE infusion is safe and about two orders of magnitude more antithrombotic than enoxaparin infusion in the baboon model of thrombosis.

Profound resolution of early atherosclerosis with conjugated linoleic acid

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid and has been shown to suppress the development of atherosclerosis in experimental models. However, the mechanism involved is unclear although it is believed it may act as a cyclooxygenase inhibitor or as an agonist of the nuclear receptors, peroxisome proliferator activated receptors (PPARs). In this study, we examined the effect of cis-9, trans-11: trans-10, cis-12-CLA (80:20 blend) on the regression of pre-established atherosclerosis. ApoE −/− mice fed a 1% cholesterol diet were randomized at 8 weeks to continue receiving the diet supplemented with 1% control saturated fat or 1% CLA blend for a further 8 weeks. CLA supplementation did not simply prevent progression but induced almost complete resolution of atherosclerosis. Although CLA inhibited platelet deposition, as detected by staining of platelet glycoprotein α 11bβ 111a, it did not inhibit COX-mediated generation of prostaglandins in this model. However, PPARα and PPARγ expression was increased in the aorta of the CLA-treated animals. This was coincident with decreased macrophage accumulation and decreased expression of the macrophage scavenger receptor CD36 and increased apoptosis in the aorta in vivo. CLA induces the resolution of atherosclerosis by negatively regulating the expression of pro-inflammatory genes and inducing apoptosis in the atherosclerotic lesion.

In Vitro Verification of Antithrombotic Effect of Recombinant Soluble Nucleotide Triphosphate Diphosphohydrolase 1

Ecto-ADPase (NTPDase1 or CD39) has been identified on endothelial cells and found to be antithrombogenic, with actions resulting from degradation of adenosine diphosphate (ADP), a thrombogenic molecule secreted by activated platelets at sites of vascular injury. Reasoning that the ADPase activity of CD39 might provide clinical use as an antithrombotic agent, the authors investigate the comparative ability of the agent to inhibit platelet and fibrin deposition. With use of an in vitro perfusion system, fresh, heparinized human blood was passed over expanded polytetrafluoroethylene grafts at hemodynamic conditions similar to those observed in the human arterial circulation. Three different concentrations of CD39 (30 mcg/mL, 100 mcg/mL, and 300 mcg/mL) were compared with abciximab (4 mcg/mL) and heparin controls. The deposition of radiolabeled platelets and fibrinogen was measured in five perfusions for each treatment group. The addition of soluble CD39 to heparinized blood inhibited platelet deposition to an extent greater than that of heparin alone (P =.04). Effects were similar to those observed with abciximab. The addition of CD39 to heparinized blood did not result in augmentation of fibrin inhibition beyond that observed with heparin alone. The fibrin inhibitory effects of CD39 appeared to be less significant than the fibrin inhibition observed with abciximab, but this difference did not attain statistical significance. These results suggest that recombinant CD39 holds potential as antithrombotic agent, with the potential to achieve antiplatelet effects similar to that observed with abciximab.

Aurintricarboxylic Acid Attenuates Intimal Thickening after Balloon Injury of the Rabbit Aorta

Injury to the arterial wall initiates a cascade of events including platelet deposition and an increase in procoagulant activity of the vessel wall that is associated with intimal thickening and vascular wall remodeling. This study was designed to characterize the effects of aurintricarboxylic acid (ATA), an inhibitor of von Willebrand factor function, on vascular procoagulant activity and the development of intimal thickening after balloon-induced injury to the rabbit aorta. Treatment with ATA, aspirin, or the combination of agents at doses that attenuated platelet aggregation decreased platelet deposition and procoagulant activity bound to the vessel wall after injury. Treatment with ATA reduced the intimal thickening observed 2 weeks after injury. Surprisingly, aspirin treatment had no effect on intimal thickening. These data indicate that inhibition of platelet deposition, while it is able to attenuate local thrombin elaboration, is not alone sufficient to attenuate subsequent intimal thickening that occurs in response to arterial injury.

Blockade of Platelet GPIIB-IIIA (Integrin αIIbβ3) in Flowing Human Blood Leads to Passivation of Prothrombotic Surfaces

We examined the impact of platelet activation on platelet adhesion to collagen in flowing human blood. ADP activation of platelets in ex vivo flowing blood resulted in paradoxical inhibition of platelet deposition on collagen. Blockade of fibrinogen binding to platelets by Lamifiban, a competitive antagonist of GPIIb-IIIa (integrin alpha(IIb)beta(3)), reversed this inhibition, leading to a marked increase in integrin alpha(2)beta(1)-dependent platelet adhesion. Analysis of integrin alpha(2)beta(1)-dependent platelet adhesion to collagen indicated that ADP-induced suppression of platelet adhesion is the result of trans-dominant inhibition of integrin alpha(2)beta(1) caused by fibrinogen binding to integrin GPIIb-IIIa. Lamifiban blocked fibrinogen binding, reversing the trans-dominant inhibition of alpha(2)beta(1) dependent adhesion to collagen. The GPIIb-IIIa antagonist resulted in the formation of a non-thrombogenic, passivated surface comprised of an adherent platelet monolayer. This unexpected consequence of blocking fibrinogen binding to GPIIb-IIIa may explain the long-term benefits of short-term GPIIb-IIIa antagonist treatment of Acute Coronary Syndrome patients.

Inhibition of platelet-collagen interaction: an in vivo action of insulin abolished by insulin resistance in obesity.

Insulin resistance is associated with an increased risk of atherothrombotic vascular disease, but the mechanisms are poorly understood. We determined how insulin in vivo regulates platelet activation in nonobese and obese subjects by using methods mimicking thrombus formation. Twelve nonobese (aged 42+/-2 years, body mass index 24.0+/-0.4 kg/m(2)) and 14 obese (aged 43+/-1 years, body mass index 37.2+/-1.5 kg/m(2)) subjects were studied under euglycemic hyperinsulinemic (3-hour insulin infusion of 1 mU. kg(-1). min(-1)) conditions. Before and at the end of hyperinsulinemia, the following were determined: (1) platelet-related early hemostasis (shear rate of approximately 4000 s(-1)) by platelet function analysis; (2) platelet deposition to collagen during whole-blood perfusion (shear rate of 1600 s(-1)); (3) aggregation responses to collagen, thrombin receptor-activating peptide, ADP, and epinephrine; and (4) platelet cGMP concentrations. Insulin action on glucose metabolism was 69% lower in the obese subjects (1.6+/-0.2 mg. kg(-1). min(-1)) than in the nonobese subjects (5.4+/-0.4 mg. kg(-1). min(-1), P<0.0001). The in vivo insulin infusion inhibited platelet deposition to collagen from 4.3+/-0.6x10(6) to 3.5+/-0.4x10(6) per square centimeter in the nonobese subjects (P<0.05) but failed to do so in the obese subjects (5.2+/-0.8x10(6) versus 5.5+/-0.7x10(6) per square centimeter, P=NS; P<0.01 versus nonobese subjects). Epinephrine- and ADP-primed closure times by platelet function analysis were prolonged by insulin in the nonobese but not the obese subjects (P<0.05 for between-group difference). In the nonobese subjects, insulin decreased aggregation to all agonists and significantly increased platelet cGMP concentrations (2.5+/-0.3 versus 3.2+/-0.5 pmol/10(9) for before versus after insulin, respectively; P<0.01). In the obese subjects, insulin did not alter collagen-induced aggregation or cGMP concentrations (1.9+/-0.2 versus 1.8+/-0.1 pmol/10(9) for before versus the end of in vivo hyperinsulinemia, respectively; P=NS). These data demonstrate that normal in vivo insulin action inhibits platelet interaction with collagen under conditions mimicking thrombus formation and reduces aggregation to several agonists. These platelet-inhibitory actions of insulin are blunted or absent in obese subjects and could provide 1 mechanism linking insulin resistance to atherothrombotic disease.