High Shear Stress Conditions Research Articles

Commentary on ‘ABO Blood Type does not Influence the Risk of Cardiovascular Complications and Mortality After Vascular Surgery’

During the past four decades, ABO blood types has been proposed as one of the prognostication factors of cardiovascular disease and non-O blood groups have been identified as a risk factor for coronary artery disease and associated mortality. A similar trend has been described for development of cardiovascular complications in the postoperative period. These interesting observations have been attributed to elevated levels of vonWillebrand factor in individuals with non-O blood groups, which plays a critical role in subendothelial adhesion of platelets, a process that is considered an initial step in acute clot formation and atherogenesis, especially under high shear stress conditions. In this issue of European Journal of Vascular and Endovascular Surgery, Bakker and colleagues present a retrospective review of 4679 patients undergoing vascular surgery from 1990 through 2011. Cardiac complications occurred in 4.9% of non-O blood type and 5.5% of O blood group patients (P 1⁄4 0.42) within 30 days after surgery, and there was no significant difference in mortality during a median follow up of 4 years. In multivariate analysis, blood type was not identified as an independent predictor of cardiac complication after major vascular surgery. This is basically a negative study, however the absence of evidencemay not be the same as evidence of absence due to some limitations of this study, including its retrospective nature, relatively small number of patients with short follow up compared to previous studies with 24e26 year follow up, and not including all perioperative and late thrombotic events (venous thromboembolic, graft occlusion, limb ischemia) in their analysis. While the results of this study conflict in part with previous observations, the authors observed significantly higher prevalence of ischemic heart disease (p1⁄4 0.02) in the non-O group at baselinewhereas therewere strong trends in the prevalence of cerebrovascular disease (p 1⁄4 0.08) and hypertension (p1⁄4 0.06) in the O group. Multivariate regression analysis was performed adjusting for several demographic variables, as well as ischemic heart disease. Although this is an accepted statistical method to examine the blood type as an independent contributing factor, it eliminates the interaction between the blood type and the prevalence of the ischemic heart disease.

Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE−/− mice

AimsWall shear stress differentially regulates the arginase pathway in carotid arteries perfused ex vivo. Specific patterns of wall shear stress can locally determine atherosclerotic plaque size and composition in vivo. The present work investigates the effects of arginase inhibition on shear stress induced plaque composition. Methods and resultsCarotid arteries of apolipoprotein E deficient mice were exposed to high (HSS), low (LSS) and oscillatory (OSS) shear stress conditions by the placement of a local shear stress modifier device for 9 weeks with or without the administration of the arginase inhibitor N-ω-Hydroxy-nor-L-arginine (nor-Noha) (10 mg/kg, i.p., 5 days/week). Carotid arginase activity was measured by colorimetric determination of urea. Atherosclerotic plaque size and composition, arginase expression and cellular localization were assessed by immunohistochemistry. Arginase activity was significantly increased in both LSS and OSS regions as compared to HSS. In the lesions, arginase II isoform co-localized preferentially with EC. Inhibition of arginase by nor-Noha decreased arginase activity and reduced plaque size in both LSS and OSS regions. Arginase inhibition affected mainly the composition of plaques developed in LSS regions by decreasing the total vascular ROS, the number of macrophages, apoptosis rate, lipid and collagen contents. ConclusionsArginase activity is modulated by patterns of wall shear stress in vivo. Chronic inhibition of vascular arginase decreased the size of atherosclerotic lesions in both OSS and LSS regions, whereas changes on plaque composition were more pronounced in plaques induced by LSS. We identified wall shear stress as a key biomechanical regulator of arginase during plaque formation and stability.

Shear Stress Initiates Cyclophilin D-Dependent Phosphatidylserine Exposure and Integrin αIIbβ3 Cleavage in Platelets

Abstract 3301When platelets are simultaneously stimulated by multiple agonists, such as thrombin and collagen, a subpopulation of procoagulant platelets is formed that is characterized by phosphatidylserine (PS) exposure, integrin αIIbβ3 inactivation, and a rounded morphology in a process that is dependent on the mitochondrial permeability transition pore (mPTP) regulatory protein cyclophilin D (CypD). Recently, we have found that, in the absence of platelet CypD, platelet accumulation is markedly accentuated both in vitro and in vivo indicating that CypD-dependent procoagulant platelet formation may limit thrombus growth. Interestingly, the importance of CypD in limiting platelet accumulation was most evident in shear stress conditions consistent with arterial flow. In low (300 s−1) and high (1500 s−1) shear conditions, CypD null platelet accumulation was increased 1.5- and 4-fold compared to wild type platelets, respectively.Platelet activation and procoagulant platelet formation were examined in various shear conditions. In platelets subjected to increasing shear, PS exposure, but not P-selectin expression or integrin αIIbβ3 activation, was observed in a platelet subpopulation. When subjected to high shear in vitro as many as 70 % of platelets expressed high levels of PS on their surface within 5 min, and shear-dependent PS exposure was observed in as little as 30 seconds in high shear stress conditions. Previously we demonstrated that agonist-initiated PS exposure is closely associated with integrin αIIbβ3 cleavage and inactivation. In shear-treated, as in strongly-stimulated platelets, integrin αIIbβ3 cleavage was closely associated with PS exposure. PS exposure and integrin αIIbβ3 cleavage were not observed in the absence of VWF. In contrast to agonist-initiated PS exposure, shear stress-induced PS exposure was observed even in the absence of extracellular calcium.In a previous study, platelet PS exposure and apoptosis induced by ristocetin were found to be closely associated with an increase in the expression of the BH3-proteins, Bax and Bak. This finding, along with the observation that shear-stress dependent PS exposure occurred even in the absence of extracellular calcium led us to examine the role of Bax and Bak. However, shear stress-induced PS exposure was unaffected in Bax/Bak null platelets. In contrast, both shear stress-induced PS exposure and integrin αIIbβ3 cleavage were markedly blunted in CypD null platelets. Furthermore, shear stress induced-PS exposure was closely associated with loss of mitochondrial transmembrane potential, together indicating the importance of mPTP formation in the regulation of PS exposure in high shear stress conditions. These data identify a novel VWF- and CypD-dependent pathway that results in rapid PS exposure and integrin αIIbβ3 cleavage in high-shear stress conditions. This shear stress-initiated pathway of PS exposure, distinct from thrombin-collagen initiated PS exposure, does not require extracellular calcium and is not associated with P-selectin expression. Disclosures:Jobe:Baxter: Membership on an entity's Board of Directors or advisory committees.

The W–x–x–W Motif in the TSR1 of ADAMTS13 Is Important for Its Secretion

Abstract 4389 Introduction:The W-x-x-W motif is commonly found in the thrombospondin type 1 repeat (TSR) of various extra cellular proteins called TSR super family proteins, including thrombospondins, ADAMTSs, F-spondin and properdin. The W–x–x–W motif is known to bind with heparin, heparan sulfate proteoglycans, collagen and transforming growth factor-β, suggesting functional significance in cell–cell interaction and/or cellular signaling. However, the function of W-x-x-W in ADAMTS13 is unclear. In this study, we investigated the role of the W-x-x-W motif of ADAMTS13. Materials and Methods:We generated a W-x-x-W mutant (W387A) construct of ADAMTS13, and expressed the mutant and the wild-type constructs in HELA cells. Percentage of the protein secretion was defined as the concentration in the culture medium divided by the concentrations in the culture medium and cell lysates, multiplied by 100%. The binding affinity of the mutant or wild-type ADAMTS13 was investigated by enzyme-linked immunosorbent assay. Measurement of ADAMTS13 proteolytic activity toward von Willebrand factor (VWF) multimers was based on the generation of a dimeric 176-kDa fragment resulting from cleavage of VWF at the Y1605-M1606 bond, under denaturing condition and high shear stress condition, analyzed by Western blots. Results:SDS-PAGE gel analysis showed that the W387A mutant was secreted less efficiently relative to the wild-type construct. As for the binding affinity for the VWF multimer, there was no difference between the wild-type and mutant ADAMTS13. The W387A mutant was less active under denaturing condition; the same result was reproduced when FRETS-VWF73 was used as the substrate. However, under high shear stress condition, the mutant was as efficient as the wild-type ADAMTS13. Conclusions:The W–x–x–W motif is necessary for efficient secretion of ADATMS13. Further studies are needed to determine the contribution of the motif to the VWF cleave activity of ADAMTS13. Disclosures:No relevant conflicts of interest to declare.

ADAMTS13 Improving the Cell Engraftment Efficacy in Mouse Model of Bone Marrow Transplantation

AbstractAbstract 1077The adhesive protein von Willebrand factor (VWF) plays an essential role on physiologic hemostasis, mediating platelet aggregation under high shear stress conditions. However excessive functions of VWF could cause thrombotic occlusion of microvasculature such as arterial capillaries, where blood flow creates a typical high shear stress. The VWF-cleaving protease ADAMTS13 is therefore thought to down-regulate precisely the VWF function to maintain enriched microcirculation. In this context, we hypothesized that this ADAMTS13 role might contribute to better donor cell homing and engraftment in various cell therapy approaches, in which fluent blood flow could be critical in the microcirculation system. To test this hypothesis, we investigated the essential role of ADAMTS13 on the donor cell engraftment using bone marrow transplantation (BMT) model in Adamts13 −/− and wild-type mice. Irradiated recipient mice were received 2 × 106 GFP positive cells from the sex-matched GFP donor mice. All of irradiated recipient mice without receiving BMT died within 21 days. Although there is no difference between Adamts13 −/− and wild-type mice in survival rate after 7days of BMT, Kaplan-Meier analysis revealed that the percent ratio of survival rate starts significantly declining after 14 days of BMT in the group of Adamts13 −/− mice. The successful cell engraftment in BMT was assessed by the number of GFP-positive neutrophils in peripheral blood at the several time points from BMT. As a result, the duration achieving the number of GFP-positive neutrophils over 500/μL was found to significantly delay in the Adamts13 −/− mice, as compared with the wild-type mice (20.2±3.8 days vs. 14.4±3.3 days). However, histological examination during the whole observation periods could not detect any typical thrombotic lesions of micro vessels developed in both wild-type and Adamts13 −/− mice. In addition, the delayed cell engraftment observed in the Adamts13 −/− mice became normalized by the bolus administration of recombinant ADAMTS13 (10 μg/mouse) at the day 0 of BMT. Bone marrow analysis at the day 1 of BMT revealed that the number of GFP-positive blood cells in bone marrow was significantly reduced in the Adamts13 −/− mice as compared with the wild-type mice, which could result in the delayed cell expansion at the day 7 and day14 of BMT in Adamts13 −/− mice. The single bolus injection of recombinant ADAMTS13 was found to fully correct the delayed cell expansion in bone marrow in the Adamts13 −/− mice. Our results indicate that the regulation of VWF-mediated thrombotic or inflammatory responses by ADAMTS13 could contribute to better microcirculation which could be critical for efficient donor cell homing and engraftment in BMT, suggesting a therapeutic potential of ADAMTS13 in cell therapy approaches. Disclosures:Soejima:The Chemo-Sero-Therapeutic Research Institute: Employment.

Infection of Vascular Endothelial Cells with Human Cytomegalovirus under Fluid Shear Stress Reveals Preferential Entry and Spread of Virus in Flow Conditions Simulating Atheroprone Regions of the Artery

Atherosclerosis is a major pathogenic factor in cardiovascular diseases, which are the leading cause of mortality in developed countries. While risk factors for atherosclerosis tend to be systemic, the distribution of atherosclerotic plaques within the vasculature is preferentially located at branch points and curves where blood flow is disturbed and shear stress is low. It is now widely accepted that hemodynamic factors can modulate endothelial gene expression and function and influence the pathophysiological changes associated with atherosclerosis. Human cytomegalovirus (HCMV), a ubiquitous pathogen, has long been proposed as a risk factor for atherosclerosis. To date, the role of HCMV in atherogenesis has been explored only in static conditions, and it is not known how HCMV infection is influenced by the physiological context of flow. In this study, we utilized a parallel-plate flow system to simulate the effects of shear stresses in different regions of the vasculature in vitro. We found that endothelial cells cultured under low shear stress, which simulates the flow condition of atheroprone regions in vivo, are more permissive to HCMV infection than cells experiencing high shear stress or static conditions. Cells exposed to low shear stress show increased entry of HCMV compared to cells exposed to high shear stress or static conditions. Viral structural gene expression, viral titers, and viral spread are also enhanced in endothelial cells exposed to low shear stress. These results suggest that hemodynamic factors modulate HCMV infection of endothelial cells, thus providing new insights into the induction/acceleration of atherosclerosis by HCMV.

Abstract 208: Laminar Shear Stress Decreases Endothelial Microparticles Release by a Nitric Oxide- and ABCA1-Dependent Mechanism

Microparticles (MP) are shed-membrane sub-micron vesicles released upon cell activation or apoptosis. Their formation is associated with loss of cell membrane asymmetry and exposure of phosphatidylserine (PS), resulting from increases in floppase activities such as ABCA1. Plasma endothelial MP (EMP) are surrogate markers of endothelial dysfunction and their levels increase in patients with cardiovascular diseases. However, the mechanisms governing EMP release in vivo remain largely unknown. As nitric oxide (NO) is a key regulator of the vasoprotective effect of laminar shear stress (SS) on the endothelium, we tested the hypothesis that endothelial NO regulates EMP release under different SS profiles. Shed-annexinV+ EMPs were analyzed by flow cytometry in the medium of confluent HUVECs (passage 1-3) subjected to either high- (15 dyne/cm2; atheroprotective) or low- (1.5 dyne/cm2; atheroprone) SS conditions for 24 hrs. HUVECs exposed to high-SS released 3-fold less EMPs compared to low-SS conditions (326±37 vs. 130±23AnnV+MPs/μL, p<0.001). High-SS EMPs also externalized less PS, as estimated by the mean fluorescence of FITC-AnnV labelling (15.6±6.1 vs. 24.2±9.4 AU, p<0.001). Exposure to L-NAME (10-5M), but not D-NAME, increased high-SS-induced EMP release by over 3-fold (p<0.001) and the NO donor SNAP (10-5M) significantly decreased low-SS-induced EMP formation by 50% (p=0.05). In addition, L-NAME increased the mean fluorescence of EMP labelling under high SS (p<0.05), whereas SNAP decreased it under low SS (p<0.05). We then evaluated ABCA1 expression under these conditions. high-SS significantly decreased the expression of ABCA1 both at the mRNA and protein level by 2- and 20-fold, respectively (p<0.05 vs low SS). L-NAME augmented ABCA1 mRNA level in cells exposed to high SS (p<0.05), whereas SNAP significantly decreased ABCA1 mRNA level under low SS conditions (p<0.05). Finally, the LXRα agonist TO90131 (0.1mM) not only increased endothelial ABCA1 expression by 20-fold, but also augmented MP level under high SS conditions. Altogether, these results demonstrate that atheroprotective shear stress limits the release of procoagulant endothelial MP through a NO-dependent regulation of endothelial ABCA1 expression.

New Insights into von Willebrand Disease and Platelet Function

Regulation of binding between von Willebrand factor (VWF) and the platelet receptor glycoprotein (GP) Ibα is one of the key steps in controlling hemostasis and thrombosis. On vascular injury at sites of high shear rates, the GPIbα interaction with subendothelial-bound VWF will initiate the tethering of circulating platelets to the vessel wall. Tethered platelets subsequently roll on the damaged vessel wall, a process that is amplified by the activation of the platelet integrin αΙΙbβ3 (GPIIb/IIIa). The initial tethering to VWF is rapidly followed by platelet binding to collagen through specific receptors (GPVI and α2β1), leading to firm adhesion, activation, and additional stable bonds mediated by αΙΙbβ3. The above described interactions can result in two distinct processes: physiological hemostasis and pathological thrombosis. Furthermore, VWF carries coagulation factor VIII, which is involved in thrombin formation that in addition to activating platelets, mediates fibrin formation and has several other actions. The importance of VWF in hemostasis is well known in patients suffering from von Willebrand disease (VWD) who present with a defect in both platelet plug and fibrin formation. Type 2B VWD is of special interest as it may provide further insight into the mechanism by which VWF promotes the adhesion of platelets to a thrombogenic surface under conditions of high shear stress. The variant phenotypic manifestations in patients affected with type 2B VWD, however, have raised the question of locus heterogeneity in VWD as a consequence of, for example, additional defects in receptor or signaling proteins mediating platelet adhesion and aggregation. Indeed, quite a few polymorphisms of platelet receptors have been associated with increased bleeding in VWD. However, many aspects of the disease remain to be elucidated. For instance, thrombin and platelet procoagulant activity may be important counterplayers to determine the severity of the bleeding complications associated with VWD.

Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation

Interactions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus.

The Role of Platelets in Atherothrombosis

Platelets have evolved highly specialized adhesion mechanisms that enable cell-matrix and cell-cell interactions throughout the entire vasculature irrespective of the prevailing hemodynamic conditions. This unique property of platelets is critical for their ability to arrest bleeding and promote vessel repair. Platelet adhesion under conditions of high shear stress, as occurs in stenotic atherosclerotic arteries, is central to the development of arterial thrombosis; therefore, precise control of platelet adhesion must occur to maintain blood fluidity and to prevent thrombotic or hemorrhagic complications. Whereas the central role of platelets in hemostasis and thrombosis has long been recognized and well defined, there is now a major body of evidence supporting an important proinflammatory function for platelets that is linked to host defense and a variety of autoimmune and inflammatory diseases. In the context of the vasculature, experimental evidence indicates that the proinflammatory function of platelets can regulate various aspects of the atherosclerotic process, including its initiation and propagation. The mechanisms underlying the proatherogenic function of platelets are increasingly well defined and involve specific adhesive interactions between platelets and endothelial cells at atherosclerotic-prone sites, leading to the enhanced recruitment and activation of leukocytes. Through the release of chemokines, proinflammatory molecules, and other biological response modulators, the interaction among platelets, endothelial cells, and leukocytes establishes a localized inflammatory response that accelerates atherosclerosis. These inflammatory processes typically occur in regions of the vasculature experiencing low shear and perturbed blood flow, a permissive environment for leukocyte-platelet and leukocyte-endothelial interactions. Therefore, the concept has emerged that platelets are a central element of the atherothrombotic process and that future therapeutic strategies to combat this disease need to take into consideration both the prothrombotic and proinflammatory function of platelets.

Preferential weld corrosion of X65 pipeline steel in flowing brines containing carbon dioxide

The aim of this research was to investigate the cause of the severe localised corrosion that sometimes occurs at welds in carbon steel pipelines carrying hydrocarbons and inhibited brines saturated with carbon dioxide. A rotating cylinder electrode apparatus was designed so that electrodes machined from the weld metal, heat affected zone and parent material of welded X65 pipeline steel could be galvanically coupled and tested in high shear stress conditions. The galvanic currents flowing between the weld regions were recorded using zero resistance ammeters, and their self-corrosion rates were found by polarisation resistance measurements. The total corrosion rate of each weld region was obtained from the sum of the self-corrosion and galvanic contributions. In uninhibited conditions, the weld metal and heat affected zone were both cathodic to the parent material, and localised corrosion was prevented. However, when an oilfield corrosion inhibitor was present, a current reversal took place, which resulted in accelerated weld corrosion. Electrochemical impedance spectroscopy showed that the inhibitor film had lower electrical resistance and was less protective on the weld metal than on the parent material. At the highest shear stress, a second current reversal could occur when the inhibitor was removed from all regions of the weld, and there was a return to the original galvanic behaviour. It was concluded that preferential weld corrosion was caused by unstable conditions in which the inhibitor film was selectively disrupted on the weld metal but remained effective on the other weld regions.

Efficacy of Contrast-enhanced US and Magnetic Microbubbles Targeted to Vascular Cell Adhesion Molecule–1 for Molecular Imaging of Atherosclerosis

To evaluate whether microbubbles targeted to vascular cell adhesion molecule-1 (VCAM-1) (CD106) coupled with a magnetic guidance system could improve the efficacy of contrast-enhanced molecular ultrasonography (US) of atherosclerosis in the aorta. The animal research committee at Southern Medical University approved all experiments. Adherence of magnetic VCAM-1-targeted microbubbles, control inactive magnetic microbubbles, and nonmagnetic VCAM-1-targeted microbubbles to VCAM-1-Fc was determined in vitro by using a flow chamber at variable shear stress (1-24 dyne/cm(2)) under magnetic field guidance. Attachment of microbubbles under magnetic field guidance was determined in vivo with fluorescent microscopy and contrast-enhanced US of the abdominal aorta in wild-type (C57BL/6) or apolipoprotein E (APOE)-deficient mice on a regular or hypercholesterolemic diet. General factorial analysis of variance was used to compare the targeted effect of the microbubbles among different animal groups to identify significant differences. Attachment was noted for magnetic and nonmagnetic microbubbles but not for inactive magnetic microbubbles; firm attachment at high shear stress (16-20 dyne/cm(2)) was achieved only with magnetic microbubbles. Fluorescence intensity and video intensity were significantly higher in magnetic microbubbles with magnetic field guidance than in inactive magnetic microbubbles and nonmagnetic microbubbles (P < .05). Video intensity from retained magnetic microbubbles in APOE-deficient mice was significantly greater than that in wild-type mice (mean video intensity for APOE-deficient mice: 28.25 [interquartile range, or IQR, 26.55-29.20] with a hypercholesterolemic diet and 16.10 [IQR, 14.15-18.75] with a regular diet; mean video intensity for wild-type mice: 9.55 [IQR, 8.85-10.5] with a hypercholesterolemic diet and 2.90 [IQR, 1.25-3.85] with a regular diet; P < .001). Use of a magnetic targeted microbubble system results in greater attachment to endothelial VCAM-1 in atherosclerotic aortas in conditions of high shear stress and improved detection of early inflammatory changes of atherosclerosis.

High shear flow induces migration of adherent human platelets

Shear forces are generated in all parts of the vascular system and contribute directly and indirectly to vascular disease progression. Endothelial cells are able to adapt to flow conditions, and are known to polarize and migrate in response to shear forces. Platelets exposed to shear stress are activated and release bioactive molecules from their alpha granules. So far, platelets have been considered to be static cells that do not leave the site of tight adhesion. However, we have recently been able to demonstrate the capacity of platelets to migrate in response to stromal derived factor-1 (SDF-1). In this project, we have demonstrated that platelets accumulate in areas with a high concentration of SDF-1 under flow conditions and respond to high shear stress by cellular polarization, cytoskeletal reorganisation, and flow-directed migration. In this context, we have shown increased Wiskott-Aldrich Syndrome protein (WASP) phosphorylation and intracellular redistribution of focal adhesion kinase (FAK) under high-shear stress conditions. The effect of flow-induced platelet migration has not previously been recognized and offers a new role for platelets as mobile cells. Their migratory potential may enable platelets to cover intimal lesions and contribute to vascular repair.

An Ultrasound Contrast Agent Targeted to P-Selectin Detects Activated Platelets at Supra-Arterial Shear Flow Conditions

To evaluate targeting of a microbubble contrast agent to platelets under high shear flow using the natural selectin ligand sialyl Lewis. Biotinylated polyacrylamide Sialyl Lewis or biotinylated carbohydrate-free polymer (used as a control) were attached to biotinylated microbubbles via a streptavidin linker. Activated human platelets were isolated and attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets or platelet dishes blocked by an anti-P-selectin antibody served as negative control substrates. Dishes coated by recombinant P-selectin served as a positive control substrate. Microbubble adhesion was assessed by microscopy in an inverted parallel plate flow chamber, with wall shear stress values of 40, 30, 20, 10, and 5 dynes/cm2. The ratio of binding and passing microbubbles was defined as capture efficiency. There was no significant difference between the groups regarding the number of microbubbles in the fluid flow at each shear rate. Sialyl Lewis-targeted microbubbles were binding and slowly rolling on the surface of activated platelets and P-selectin-coated dishes at all the flow conditions including 40 dynes/cm2. Capture efficiency of targeted microbubbles to activated platelets and recombinant P-selectin decreased with increasing shear flow: at 5 dynes/cm2, capture efficiency was 16.11% on activated platelets versus 21.83% on P-selectin, and, at 40 dynes/cm2, adhesion efficiency was still 3.4% in both groups. There was neither significant adhesion of Sialyl Lewis-targeted microbubbles to control substrates, nor adhesion of control microbubbles to activated platelets or to recombinant P-selectin. Microbubble targeting using sialyl Lewis, a fast-binding ligand to P-selectin, is a promising strategy for the design of ultrasound contrast binding to activated platelets under high shear stress conditions.

Aspirin as antiplatelet agent in diabetes. PROS-

Macrovascular complications in diabetes mellitus (DM) manifest themselves as accelerated atherosclerosis, clinically resulting in premature coronary artery disease (CAD), increased risk of cerebrovascular disease, and severe peripheral vascular disease [1]. Patients with type 2 diabetesmellitus (T2DM) have a twoto four-fold increase in the risk of CAD and patients with DM but without previous myocardial infarction (MI) carry the same level of risk for subsequent acute coronary events as non-diabetic patients with previous MI [2]. The abnormal metabolic state that accompanies diabetes is responsible for abnormalities in endothelial and platelet function, which may contribute to the cellular events that cause atherosclerosis and subsequently increase the risk of the adverse cardiovascular events. In particular, an altered platelet metabolism and changes in intraplatelet signalling pathways may contribute to the pathogenesis of atherothrombotic complications of diabetes. It is still under debate whether enhanced platelet activation is merely a consequence of more prevalent atherosclerotic lesions (relevant to a risk of thrombosis complicating plaque rupture) or reflects the influence of the accompanyingmetabolic disturbances on platelet biochemistry and function [3]. Since 1965 many studies demonstrated a variety of platelet alterations in diabetes [4]. Chronic hyperglycemia has been clearly identified as a causal factor for in vivo platelet activation in DM patients [3,5]. In 1990, Davi et al. demonstrated enhanced thromboxane (TX) biosynthesis in T2DM and provided evidence for its platelet origin [6]. Tightmetabolic control led to a reduction of TX levels in the same study [6]. We subsequently reported that the release of a platelet-derived inflammatorymolecule, soluble CD40 ligand, able to activate platelets, is largely thromboxane dependent, thus suggesting a potential mechanismof amplificationof thromboxane-dependent platelet activation [7]. Interestingly, the metabolic disorder rather than the attendant vasculardiseaseappears tobe responsible forpersistentplatelet activation in this setting [3]. This important issue has been further proven by the detection of enhanced TX biosynthesis in type 1 diabetic children and adolescents at diagnosis, presumably driven by the underlying inflammatory burden [8], as well as in newly-diagnosed type 2 diabetic subjects free of vascular disease. In the latter the biochemical abnormality is reversible by dampening postprandial glycemic spikes with acarbose treatment [9]. Consistently, acute, short-term hyperglycemia induces increased activation of platelets exposed to high shear stress conditions

STRUCTURAL EXPERIMENT OF REINFORCED BEAMS USING STRAIN-HARDENING CEMENTITIOUS COMPOSITES

In these years, high-rise apartment buildings with core wall structural system have been constructed. Since span of coupling beams used in the core wall system is too short, large drift angle will be developed. However, RC short span beams do not have excellent ductility under high shear stress condition during earthquake. To resolve this problem, it was decided to use high-performance fiber reinforced cement composite SHCC (Strain-Hardening Cementitious Composites), which exhibits excellent ductility, for short-span beams. In this study, shear bending tests under antisymmetrical moment condition of SHCC beam specimens were carried out to investigate their structural performances. The accuracy of the calculated flexural strength and shear strength values was also examined.

Glycoprotein Ibalpha Inhibitor Complex Crystal Structure at High Resolution.

Abstract 774 Introduction:The platelet Glycoprotein Ib/V/IX (GpIb/V/IX) complex is considered a major target for anticoagulant therapy. The primary function of the receptor is to mediate platelet adhesion to von Willebrand factor (VWF) bound to damaged sub-endothelium. This represents the first critical step for platelet adhesion under conditions of high fluid shear stress. GpIb/V/IX is implicated in a number of thrombotic pathological processes such as stroke or myocardial infarction and the bleeding disorders Bernard-Soulier syndrome, platelet type von Willebrand disease (Pt-VWD) and thrombotic thrombocytopenic purpura. We have successfully determined the structure of the GpIbalpha N-terminal domain in complex with a potent (sub nM) 11meric peptide inhibitor (OS1) of the interaction with VWF. Methods:We have determined the crystal structure to 1.8Å resolution using molecular replacement. Results. The peptide sequence CTERMALHNLC was readily identifiable bound to GpIbalpha between the extended regulatory (R) loop and the concave surface of the leucine rich repeats. The peptide adopts one and a half turns of an alpha-helix and contacts three subsites (S1, S2 and S3). S1 and S2 reside within the leucine rich repeats and S3 has a unique feature as this subsite involves contact with the regulatory R-loop stabilizing it in a well defined conformation with helical character. This loop alters conformation between an extended beta-hairpin in the VWF-A1 bound structure and a more compact largely disordered structure in the unliganded structure. In this regard, the Pt-VWD mutations of GpIbalpha, G233V and M239V, which reside in the R-loop act by inducing a beta-conformation and thus result in a high affinity form of the receptor. Conclusions:These studies provide a strategy for targeting the GpIbalpha-VWF interaction using small molecules or alpha-helical peptides exploiting the GpIbalpha subsites described here and acting allosterically to stabilise a low affinity conformation of the receptor with an alpha helical R-loop. Ligand mimetic peptide complex crystal structures for the platelet receptors integrin aIIbb3 with RGD, and alpha2beta1 with a collagen peptide have been described and the former are currently in therapeutic use for treatment of thromboemboletic disorders. Targeting the GpIbalpha-VWF interaction may provide anti-thrombotic drugs which affect platelet adhesion under high shear stress without compromising normal processes of platelet adhesion and aggregation which may be required for normal hemostasis to function. Targeting protein-protein interactions is considered one of the great contemporary challenges in drug discovery. The understanding of how the S1S2S3 subsites provide very effective inhibition of a large protein-protein interaction has wide applicability. LRR proteins are an extended family mediating protein-protein interactions involved in a variety of disease processes such as sepsis, asthma, immunodeficiencies, atherosclerosis, alzheimers (leucine rich repeat kinase) and leukaemia (leucine rich repeat phosphatase). The structural fit of the helical curvature of the peptide with the arc of the leucine rich repeats may provide a basis for further development of alpha-helical peptide mimetics targeting other members of the LRR family which utilize the concave face. Disclosures:No relevant conflicts of interest to declare.

Thrombospondin-1 and ADAMTS13 Competitive Binding with VWF Mainly in Both A2 and A3 Domain.

Abstract 4008Poster Board III-944 IntroductionThrombospondin-1 (TSP1) is one multifunctional glycoprotein belonging to the thrombospondins family. It is mainly synthesized by megakaryocytes, stored in the intermediate zone of platelet -granules, and secreted by platelets upon thrombin activation. TSP1 has been postulated to play an important role in hemostasis for a long time. Recently, in a TSP1 knockout mouse, study showed that TSP1 protected unfolded endothelium-bound and subendothelial VWF from degradation by its plasma protease ADAMTS13, however, concrete mechanism that how to inhitbit ADAMTS13 activity by TSP1 was unclear. Materials and Methodswe constructed and expressed different domains of human VWF, mainly including D'D3, A1, A2, A3 domain, full-length VWF and ADAMTS13, then investigated the binding effect of TSP1 or ADAMTS13 with these different domains and full-length VWF by enzyme linked immunosorbent assay. We also observed the direct inhibition effect of different concentrations TSP1 on ADAMTS13 activity by Western blot method and residual-collagen binding assay (R-CBA) in denaturing condition and high shear stress condition ResultsWe found that ADAMTS13 could interact with the A1, A2, A3 domain and full-length VWF, and TSP1 could bind with the A3 domain. Meanwhile, we observed that TSP1 also had significant interaction with the A2 domain. And TSP1 competed with ADAMTS13 mainly existing in both A2 and A3 domain. The results of Western blot and R-CBA also showed that TSP1 dramatically restrained ADAMTS13 activity, especially at high concentrations, and the inhibitory effect could add up to 50%. ConclusionsOur study showed that TSP1 played competitive inhibitor role during ADAMTS13 binding and cleaving VWF, and the competitive process maybe mainly exist in both A2 and A3 domain. Disclosures:No relevant conflicts of interest to declare.

P-selectin antagonism reduces thrombus formation in humans.

Interaction of P-selectin with its glycoprotein ligand (P-selectin glycoprotein ligand type 1) mediates inflammatory processes that may also include vascular thrombosis. Platelet P-selectin expression is increased in patients with coronary heart disease, and its antagonism represents a potential future therapeutic target for the prevention and treatment of atherothrombosis. To investigate the effects of the novel small molecule P-selectin antagonist PSI-697 on thrombus formation in humans. In a double-blind randomized crossover study, thrombus formation was measured in 12 healthy volunteers, using the Badimon ex vivo perfusion chamber under conditions of low and high shear stress. Saline placebo, low-dose (2 m) and high-dose (20 m) PSI-697 and the glycoprotein IIb-IIIa receptor antagonist tirofiban (50 ng mL(-1)) were administered into the extracorporeal circuit prior to the perfusion chamber. As compared with saline placebo, blockade of platelet glycoprotein IIb-IIIa receptor with tirofiban produced 28% and 56% reductions in thrombus formation in the low-shear and high-shear chambers, respectively. PSI-697 caused a dose-dependent, but more modest, reduction in thrombus formation. Low-dose PSI-796 (2 m) reduced total thrombus area by 14% (P = 0.04) and 30% (P = 0.0002) in the low-shear and high-shear chambers, respectively. At the high dose (20 m), PSI-697 reduced total thrombus area by 18% (P = 0.0094) and 41% (P = 0.0008) in the low-shear and high-shear chambers, respectively. P-selectin antagonism with PSI-697 reduces ex vivo thrombus formation in humans. These findings provide further evidence that P-selectin antagonism may be a potential target for the prevention and treatment of cardiovascular disease.

Identification of a VWF peptide antagonist that blocks platelet adhesion under high shear conditions by selectively inhibiting the VWF-collagen interaction.

Because the collagen-VWF-GPIb/IX/V axis plays an important role in thrombus formation, it represents a promising target for development of new antithrombotic agents. We used phage display to identify potential small peptides that interfere with the VWF-collagen binding and might serve as lead products for the development of possible oral antithrombotic compounds. A random linear heptamer peptide library was used to select VWF-binding peptides. We identified a phage clone, displaying the YDPWTPS sequence, further referred to as L7-phage, that bound to VWF in a specific and a dose-dependent manner. This L7-phage specifically inhibited the VWF-collagen interaction under both static and flow conditions. Epitope mapping using deletion mutants of VWF revealed that the L7-phage does not bind to the known collagen-binding A3 domain within VWF, but to the more carboxyterminal situated C domain. This inhibition was not due to steric hindrance of the A3 domain-collagen interaction by the L7-phage. Indeed, a tetrabranched multi-antigen peptide (MAP) presenting four copies of the peptide, but not the scrambled MAP, also inhibited VWF-collagen interaction under conditions of high shear stress at a concentration of 148 nmol L(-1). Based on these results, we conclude that we have identified the first peptide antagonist that binds to the VWF C domain and by this specifically inhibits the VWF binding to collagen, suppressing platelet adhesion and aggregation under high shear conditions. As a consequence, this peptide and its future derivates are potentially interesting antithrombotic agents.