Repetitive Hypershear Activates and Sensitizes Platelets in a Dose-Dependent Manner.

Implantation of mechanical circulatory support (MCS) devices-ventricular assist devices and the total artificial heart-has emerged as a vital therapy for advanced and end-stage heart failure. Unfortunately, MCS patients face the requirement of life-long antiplatelet and anticoagulant therapy to combat thrombotic complications resulting from the dynamic and supraphysiologic shear stress conditions associated with such devices, whose effect on platelet activation is poorly understood. We developed a syringe-capillary viscometer-the "platelet hammer"-that repeatedly exposed platelets to average shear stresses up to 1000 dyne/cm(2) for as short as 25 ms. Platelet activation state was measured using a modified prothrombinase assay, with morphological changes analyzed using scanning electron microscopy. We observed an increase in platelet activation state and post-high shear platelet activation rate, or sensitization, with an increase in stress accumulation (SA), the product of shear stress and exposure time. A significant increase in platelet activation state was observed beyond an SA of 1500 dyne-s/cm(2) , with a marked increase in pseudopod length visible beyond an SA of 1000 dyne-s/cm(2) . Utility of the platelet hammer extends to studies of other shear-dependent pathologies, and may assist development of approaches to enhance the safety and effectiveness of MCS devices and objective antithrombotic pharmacotherapy management.

Comparative efficacy of in vitro and in vivo metabolized aspirin in the DeBakey ventricular assist device.

Ventricular assist devices (VADs) are implanted in patients with end-stage heart failure to provide both short- and long-term hemodynamic support. Unfortunately, bleeding and thromboembolic complications due to the severely disturbed, dynamic flow conditions generated within these devices require complex, long-term antiplatelet and anticoagulant therapy. While several studies have examined the effectiveness of one such agent, aspirin, under flow conditions, data comparing the efficacy of in vitro and in vivo metabolized aspirin is lacking. Two sets of studies were conducted in vitro with purified human platelets circulating for 30 min in a flow loop containing the DeBakey VAD (MicroMed Cardiovascular, Houston, TX, USA): (a) 20 µM aspirin was added exogenously in vitro to platelets isolated from aspirin-free subjects, and (b) platelets were obtained from donors 2 h (n = 14) and 20 h (n = 13) after ingestion of 1,000 mg aspirin. Near real-time platelet activation state (PAS) was measured with a modified prothrombinase-based assay. Platelets exposed to aspirin in vitro and in vivo (metabolized) showed 28.2 and 25.3 % reduction in platelet activation rate, respectively, compared to untreated controls. Our results demonstrate that in vitro treatment with antiplatelet drugs such as aspirin is as effective as in vivo metabolized aspirin in testing the effect of reducing shear-induced platelet activation in the VAD. Using the PAS assay provides a practical in vitro alternative to in vivo testing of antiplatelet efficacy, as well as for testing the thrombogenic performance of devices during their research and development.

Targeting gC1qR domains for therapy against infection and inflammation.

Abstract The receptor for the globular heads of C1q, gC1qR/p33, is a widely expressed cellular protein, which binds to diverse ligands including plasma proteins, cellular proteins, and microbial ligands. In addition to C1q, gC1qR also binds high molecular weight kininogen (HK), which also has two other cell surface sites, namely, cytokeratin 1 and urokinase plasminogen activator receptor (uPAR). On endothelial cells (ECs), the three molecules form two closely associated bimolecular complexes of gC1qR/cytokeratin 1 and uPAR/cytokeratin 1. However, by virtue of its high affinity for HK, gC1qR plays a central role in the assembly of the kallikrein-kinin system, leading to the generation of bradykinin (BK). BK in turn is largely responsible for the vascular leakage and associated inflammation seen in angioedema patients. Therefore, blockade of gC1qR by inhibitory peptides or antibodies may not only prevent the generation of BK but also reduce Clq-induced or microbial-ligand-induced inflammatory responses. Employing synthetic peptides and gClqR deletion mutants, we confirmed previously predicted sites for C1q (residues 75-96) and HK (residues 204-218) and identified additional sites for both C1q and HK (residues 190-202), for C1q (residues 144-162), and for HIV-1 gp41 (residues 174-180). With the exception of residues 75-96, which is located in the alphaA coiled-coil N-terminal segment, most of the identified residues form part of the highly charged loops connecting the various beta-strands in the crystal structure. Taken together, the data support the notion that gC1qR could serve as a novel molecular target for the design of antibody-based and/or peptide-based therapy to attenuate acute and/or chronic inflammation associated with vascular leakage and infection.

Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice.

Ectopic delivery of factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by concentrating the FVIIIa/FIXa enzyme-cofactor complex onto activated platelet membranes. We utilized a core rat platelet factor 4 (PF4) promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of a haemophilia A mouse (rPF4/hBDD/FVIII-/-). Platelets from rPF4/hBDD/FVIII(-/-) mice contained approximately 122 mU FVIII:C/1 x 10(9) platelets/ml with no detectable plasmatic FVIII:C, and with no effect on alpha-granule-derived platelet factor V/Va function. Paired tenase assays (+/- thrombin) confirmed that platelet (pt) FVIII (unlike platelet FV) required thrombin cleavage for complete activation. rPF4/hBDD/FVIII(-/-) mice exposed to a thrombocytotic stimulus (thrombopoietin, TPO) demonstrated a statistically-significant 66% reduction in molar ptFVIII activity with a non-significant reduction in total ptFVIII biomass. Decreased molar ptFVIII concentration correlated with loss of phenotypic correction as evaluated using a haemostatic tail-snip assay. Comparative studies using a transgenic mouse expressing human amyloid-beta-precursor protein (hAbetaPP) from the rPF4 promoter confirmed diminished hAbetaPP expression without affecting endogenous alpha-granule PF4, establishing generalizability of these observations. While Mk/platelet-released ptFVIII (unlike pFV) is proteolytically inactive, we also conclude that thrombocytotic stimuli negatively affect ptFVIII bioavailability and phenotypic efficacy, results which correlate best with molar ptFVIII concentration, and not systemically available ptFVIII.

Positive feedbacks of coagulation: their role in threshold regulation.

Tissue factor (TF), the initiator of coagulation, continuously circulates in the plasma, and the clotting system "idles," generating very low levels of active clotting enzymes, clotting products, and by-products. Given the enormous amplification potential of the clotting cascade, rigorous control is required to ensure that such low-level stimulation does not cause massive system amplification and response. We propose that among the various mechanisms of regulation, activation thresholds may play a major role. These arise when positive-feedback reactions, of which there are several in the clotting system, are regulated by inhibitors. Such thresholds act like switches, so that small stimuli and/or nonproductive local conditions will generate no response, whereas larger stimuli or the existence of local prothrombotic conditions will produce a full, explosive response. We review here the evidence for system idling, the structures of the various feedback mechanisms of clotting, the mechanisms by which they can produce threshold behavior, and the possible role of thresholds in system regulation.

Platelet factor V supports hemostasis in a patient with an acquired factor V inhibitor, as shown by prothrombinase and tenase assays.

A woman with gross hematuria was shown to have a severe isolated factor V deficiency due to a factor V inhibitor of 200 U/ml titer. Hematuria persisted despite multiple infusions of plasma but, after one transfusion with 1 U platelets, urine red blood cells decreased by more than 98%. To evaluate the patient's platelet function we performed prothrombinase and tenase assays with platelets from the patient and from normal donors. By prothrombinase assay, ionophore-activated patient platelets showed 42% of the activity of normal platelets in their ability to support prothrombin activation by activated factor X; whereas in a 'tenase' assay, which measures the platelets' ability to support factor X activation by activated factor IX + activated factor VIII, their activity was 117% of normal. The addition of excess bovine activated factor V to the prothrombinase assay fully corrected the defect. The results demonstrate the benefit of platelet transfusion and indicate that in this case the platelets are the primary source of factor V for hemostasis.

gC1qR/p33 serves as a molecular bridge between the complement and contact activation systems and is an important catalyst in inflammation.

The receptor for the globular heads of C1q, gC1qR/p33, is a ubiquitously expressed protein, which is distributed both intracellularly and on the cell-surface protein. In addition to C1q, this molecule also is able to bind several other biologically important plasma ligands, including high-molecular-weight kininogen (HK), factor XII (FXII), and multimeric vitronectin. Previous studies have shown that incubation of FXII, prekallikrein, and HK with gC1qR leads to a zinc-dependent and FXII-dependent conversion of prekallikrein to kallikrein, a requisite for kinin generation. In addition, these studies showed that normal plasma, but not plasma deficient in FXII, PK, or HK, activate upon binding to endothelial cells (EC), and that this activation could be inhibited by antibody to gClqR. In these studies, we show that incubation of serum with microtiter plate bound gC1qR results in complement activation, as evidenced by the binding and activation of C1 and generation of C4d. However, neither Clq-deficient serum nor a truncated form of gC1qR (gC1qRA74-96), supported complement activation. Taken together, the data strongly suggest that at sites of inflammation, such as vasculitis and atherosclerosis, where gC1qR as well as its two important plasma ligands, C1q and HK, have been shown to be simultaneously present, soluble or cell-surface-expressed gC1qR may contribute to the inflammatory process by modulating complement activation, kinin generation, and perhaps even initiation of clotting via the contact system. Based on these and other published data, we propose a model of inflammation in which atherogenic factors (e.g., immune complexes, virus, or bacteria) are perceived not only to convert the endothelium into a procoagulant and proinflammatory surface, but also to induce enhanced expression of cell surface molecules such as gC1qR. Enhanced expression of gC1qR in turn leads to: (i) high-affinity C1q binding and cell production of proinflammatory factors, and (ii) high-affinity HK binding and facilitation of the assembly of contact activation proteins leading to generation of bradykinin and possibly coagulation through activation of FXI.

Thrombogenic performance of a st. Jude bileaflet mechanical heart valve in a sheep model.

The sheep model is preferred for chronic evaluation of prosthetic heart valves, surgical techniques, and endocardiographic studies. A bileaflet mechanical heart valve (MHV) was implanted into a sheep model to study its in vivo performance and to evaluate the thrombogenic potential of the valve. Transesophageal echocardiography and transcranial Doppler ultrasonography measurements were conducted before and after the valve implantation. Platelet activity state (PAS) assay measurements were also conducted before and after the implantation surgery. After sheep euthanasia, the MHV was explanted and scanning electron microscopy (SEM) was performed on the explanted valve to examine changes to the MHV surface. Tissue blocks were taken from the sheep brain, left ventricle, aorta, spleen, and lung lobes for histological examination. Our results indicated that after the MHV implantation, more embolic signals were detected in the sheep carotid artery, increasing monotonously as a function of implantation time. Echocardiographic parameters including blood aortic velocity, transvalvular pressure gradient, and velocity time integral increased. PAS increased significantly after valve implantation. SEM pictures demonstrated calcium and phosphate deposition on the valve surfaces. Histological examination demonstrated hemorrhage in the lung tissue, pulmonary thrombosis, and osteogenesis in heart tissue.

Differences between mainstream and sidestream cigarette smoke extracts and nicotine in the activation of platelets under static and flow conditions.

BACKGROUND: Cigarette smoke is a primary risk factor for cardiovascular diseases. Enhanced function of the hemostatic system, in which platelets play a major role, is a significant underlying mechanism in cardiovascular disease and its progression. Epidemiological studies, complemented by physiological and biochemical data, show that cigarette smoke adversely affects platelet function, both in smokers and in nonsmokers exposed to sidestream smoke. METHODS AND RESULTS: The thrombogenic potential of platelets subjected to mainstream smoke extracts, sidestream extracts, and nicotine was measured in vitro under static and dynamic flow conditions. Platelet activation state was measured with a modified prothrombinase-based method. Mainstream and sidestream smoke extracts caused increased platelet activation. Although low-tar mainstream extracts activated platelets less than high-tar extracts, the sidestream extracts were almost equally potent. Modification of the filters of low-tar cigarettes, by blocking the air-bypass holes, raised activation rates by mainstream extracts to the level of high-tar extracts. Nicotine (50 nmol/L and 5 micromol/L) inhibited platelet activation under both flow and static conditions. CONCLUSIONS: Cigarette smoke extracts directly cause platelet activation but also markedly increase the susceptibility of platelets to activation by shear stress. In contrast, nicotine, although also a constituent of cigarette smoke, significantly reduces platelet susceptibility to shear stress.

Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential.

BACKGROUND: Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state. METHODS AND RESULTS: Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and alpha(V)beta3 integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1-deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin. CONCLUSIONS: The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction-elevated levels of PAI-1 and propensity toward thrombosis.

The platelet hammer: In vitro platelet activation under repetitive hypershear.

Mechanical circulatory support (MCS) devices, such as ventricular assist devices and the total artificial heart, have emerged as a vital therapy for advanced and end-stage heart failure. However, MCS patients face life-long antiplatelet and anticoagulant therapy to minimize thrombotic complications resulting from the dynamic and supraphysiologic device-associated shear stress conditions, whose effect on platelet activation is poorly understood. We repeatedly exposed platelets to average shear stresses up to 1000 dyne/cm(2) for as short as 25 ms in the "platelet hammer," a syringe-capillary viscometer. Platelet activation state was measured using a modified prothrombinase assay and morphological changes analyzed using scanning electron microscopy. An increase in stress accumulation (SA), the product of shear stress and exposure time, led to an increase in the platelet activation state and post-high shear platelet activation rate, or sensitization. A significant increase in platelet activation state was observed beyond an SA of 1500 dyne-s/cm(2), with a marked increase in pseudopod length visible beyond an SA of 1000 dyne-s/cm(2). The platelet hammer may be used to study other shear-dependent pathologies and may ultimately enhance the safety and effectiveness of MCS devices and objective antithrombotic pharmacotherapy management.

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus.

We have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIDelta761-1639). Productive replication of Ad/AAV/FVIIIDelta761-1639 in AAV rep-expressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIDelta761-1639). In vitro studies using Ad/AAV/FVIIIDelta761-1639 generated approximately 2-logs greater FVIII activity than mAd/FVIIIDelta761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIDelta761-1639 was injected by tail vein into three groups of hemophilia A mice (2 x 10(11) vp [n = 3]; 4 x 10(11) vp [n = 3]; 8 x 10(11) vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16%-51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1-0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (approximately 10 genomes/cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of "gutted" monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

gC1q-R/p33: structure-function predictions from the crystal structure.

Human gC1q-R (p33) is a multicompartmental cellular protein expressed on various types of cells and tissues. Although originally isolated as a receptor for C1q by virtue of its specificity for the globular heads of that molecule, a large body of evidence has now been accumulated which shows that in addition to C1q, gC1q-R can serve as a receptor for diverse ligands including proteins of the intrinsic coagulation/bradykinin forming cascade, as well as antigens of cellular, bacterial, and viral origin. Furthermore, since gC1q-R has been shown to regulate the functions of protein kinase C (PKC), it is postulated that gC1q-R-induced signaling cascade may involve activation of PKC. These data collectively therefore suggest that gC1q-R plays an important role in blood coagulation, inflammation, and infection. However, although significant progress has been made in unraveling the molecular, biochemical, and structural features of this molecule, and data in support of its biological relevance is accumulating, it is still unclear as to how the molecule is anchored on the membrane since its sequence is devoid of a classical transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor. Furthermore, while recombinant gC1q-R can bind to cell surfaces suggesting that it may bind directly to the phospholipid bilayer, our recent experiments show that, at least in vitro, gC1q-R does not bind to unilamellar vesicle preparations of either phosphatidylcholine (PC) or phosphatidylserine: phosphatidylcholine. This work was therefore undertaken to analyze the three-dimensional structure of gC1q-R in order to identify unique structural features that may serve not only to anchor the protein but also to explain its affinity for such a diversity of plasma as well as microbial and viral ligands.

Flow-induced platelet activation in mechanical heart valves.

BACKGROUND AND AIM OF THE STUDY: A study was conducted to measure in vitro the procoagulant properties of platelets induced by flow through mechanical heart valves. METHODS: The procoagulant activity of platelets was measured using a real-time assay of platelet activation state (PAS), which was based on a modification of the prothrombinase method. Acetylated prothrombin was used instead of normal prothrombin in this assay in order to eliminate the positive feedback effect of thrombin. This enabled a direct comparison between thrombin generation rates in the assay and the flow stresses that induce platelet activation. Gel-filtered platelets (10(5) per microliters) were circulated through a left ventricular assist device with two Björk-Shiley mono-leaflet mechanical heart valves mounted in opposition, and platelet activation state was measured over 30-min time courses. The results were compared with two configurations in which the leaflet motion of one of the valves was restricted (severely restricted and mildly restricted), mimicking defective function of a compromised valve in vivo, and with a control lacking valves. RESULTS: The severely restricted valve activated the platelets at a rate eight-fold higher than with unrestricted valves, and three-fold higher than with mildly restricted valves. Both restricted valves activated platelets at rates significantly higher than either the control (no valves) or the unrestricted valve. CONCLUSION: Flow through compromised mechanical heart valves causes platelet activation, which can be measured with a modified prothrombinase assay system. The ability to perform sensitive quantitative measurements in cardiovascular devices in vitro may have a significant impact on the design and development of these devices.

Evaluation of shear-induced platelet activation models under constant and dynamic shear stress loading conditions relevant to devices.

The advent of implantable blood-recirculating devices such as left ventricular assist devices and prosthetic heart valves provides a viable therapy for patients with end-stage heart failure and valvular disease. However, device-generated pathological flow patterns result in thromboembolic complications that require complex and lifelong anticoagulant therapy, which entails hemorrhagic risks and is not appropriate for certain patients. Optimizing the thrombogenic performance of such devices utilizing numerical simulations requires the development of predictive platelet activation models that account for variations in shear-loading rates characterizing blood flow through such devices. Platelets were exposed in vitro to both dynamic and constant shear stress conditions emulating those found in blood-recirculating devices in order to determine their shear-induced activation and sensitization response. Both these behaviors were found to be dependent on the shear loading rates, in addition to shear stress magnitude and exposure time. We then critically examined several current models and evaluated their predictive capabilities using these results. Shear loading rate terms were then included to account for dynamic aspects that are either ignored or partially considered by these models, and model parameters were optimized. Independent optimization for each of the two types of shear stress exposure conditions tested resulted in different sets of best-fit constants, indicating that universal optimization may not be possible. Inherent limitations of the current models require a paradigm shift from these integral-based discretized power law models to better address the dynamic conditions encountered in blood-recirculating devices.

Device thrombogenicity emulation: a novel method for optimizing mechanical circulatory support device thromboresistance.

Mechanical circulatory support (MCS) devices provide both short and long term hemodynamic support for advanced heart failure patients. Unfortunately these devices remain plagued by thromboembolic complications associated with chronic platelet activation--mandating complex, lifelong anticoagulation therapy. To address the unmet need for enhancing the thromboresistance of these devices to extend their long term use, we developed a universal predictive methodology entitled Device Thrombogenicity Emulation (DTE) that facilitates optimizing the thrombogenic performance of any MCS device--ideally to a level that may obviate the need for mandatory anticoagulation. DTE combines in silico numerical simulations with in vitro measurements by correlating device hemodynamics with platelet activity coagulation markers--before and after iterative design modifications aimed at achieving optimized thrombogenic performance. DTE proof-of-concept is demonstrated by comparing two rotary Left Ventricular Assist Devices (LVADs) (DeBakey vs HeartAssist 5, Micromed Houston, TX), the latter a version of the former following optimization of geometrical features implicated in device thrombogenicity. Cumulative stresses that may drive platelets beyond their activation threshold were calculated along multiple flow trajectories and collapsed into probability density functions (PDFs) representing the device 'thrombogenic footprint', indicating significantly reduced thrombogenicity for the optimized design. Platelet activity measurements performed in the actual pump prototypes operating under clinical conditions in circulation flow loops--before and after the optimization with the DTE methodology, show an order of magnitude lower platelet activity rate for the optimized device. The robust capability of this predictive technology--demonstrated here for attaining safe and cost-effective pre-clinical MCS thrombo-optimization--indicates its potential for reducing device thrombogenicity to a level that may significantly limit the extent of concomitant antithrombotic pharmacotherapy needed for safe clinical device use.

Thrombogenic potential of Innovia polymer valves versus Carpentier-Edwards Perimount Magna aortic bioprosthetic valves.

Trileaflet polymeric prosthetic aortic valves (AVs) produce hemodynamic characteristics akin to the natural AV and may be most suitable for applications such as transcatheter implantation and mechanical circulatory support (MCS) devices. Their success has not yet been realized due to problems of calcification, durability, and thrombosis. We address the latter by comparing the platelet activation rates (PARs) of an improved polymer valve design (Innovia LLC) made from poly(styrene-block-isobutylene-block-styrene) (SIBS) with the commercially available Carpentier-Edwards Perimount Magna Aortic Bioprosthetic Valve. We used our modified prothrombinase platelet activity state (PAS) assay and flow cytometry methods to measure platelet activation of a pair of 19 mm valves mounted inside a pulsatile Berlin left ventricular assist device (LVAD). The PAR of the polymer valve measured with the PAS assay was fivefold lower than that of the tissue valve (p = 0.005) and fourfold lower with flow cytometry measurements (p = 0.007). In vitro hydrodynamic tests showed clinically similar performance of the Innovia and Magna valves. These results demonstrate a significant improvement in thrombogenic performance of the polymer valve compared with our previous study of the former valve design and encourage further development of SIBS for use in heart valve prostheses.

Structure-function studies using deletion mutants identify domains of gC1qR/p33 as potential therapeutic targets for vascular permeability and inflammation.

The endothelial cell receptor complex for kininogen (HK) comprises gC1qR, cytokeratin 1, and urokinase-type plasminogen activator receptor and is essential for activation of the kinin system that leads to bradykinin (BK) generation. Of these, gC1qR/p33 constitutes a high affinity site for HK - the BK precursor - and is therefore critical for the assembly of the kinin-generating cascade. Previous studies have identified a putative HK site within the C-terminal domain (residues 204-218) of gC1qR recognized by mAb 74.5.2. In these studies, we used information from the crystal structure of gC1qR, to engineer several deletion (Δ) mutants and test their ability to bind and/or support BK generation. While deletion of residues 204-218 (gC1qRΔ204-218), showed significantly reduced binding to HK, BK generation was not affected when tested by a sensitive bradykinin immunoassay. In fact, all of the gC1qR deletion mutants supported BK generation with the exception of gC1qRΔ154-162 and a point mutation in which Trp 233 was substituted with Gly. Binding studies also identified the existence of two additional sites at residues 144-162 and 190-202. Moreover, binding of HK to a synthetic peptide 190-202 was inhibited by mAbs 48 and 83, but not by mAb 74.5.2. Since a single residue separates domains 190-202 and 204-218, they may be part of a highly stable HK binding pocket and therefore a potential target for drug design to prevent vascular permeability and inflammation.

High-shear stress sensitizes platelets to subsequent low-shear conditions.

Individuals with mechanical heart valve implants are plagued by flow-induced thromboembolic complications, which are undoubtedly caused by platelet activation. Flow fields in or around the affected regions involve brief exposure to pathologically high-shear stresses on the order of 100 to 1000 dyne/cm(2). Although high shear is known to activate platelets directly, their subsequent behavior is not known. We hypothesize that the post-high-shear activation behavior of platelets is particularly relevant in understanding the increased thrombotic risk associated with blood-recirculating prosthetic cardiovascular devices. Purified platelets were exposed to brief (5-40 s) periods of high-shear stress, and then exposed to longer periods (15-60 min) of low shear. Their activation state was measured using a prothrombinase-based assay. Platelets briefly exposed to an initial high-shear stress (e.g., 60 dyne/cm(2) for 40 s) activate a little, but this study shows that they are now sensitized, and when exposed to subsequent low shear stress, they activate at least 20-fold faster than platelets not initially exposed to high shear. The results show that platelets in vitro exposed beyond a threshold of high-shear stress are primed for subsequent activation under normal cardiovascular circulation conditions, and they do not recover from the initial high-shear insult.

Design optimization of a mechanical heart valve for reducing valve thrombogenicity-A case study with ATS valve.

Patients implanted with mechanical heart valves (MHV) or with ventricular assist devices that use MHV require mandatory lifelong anticoagulation for secondary stroke prevention. We recently developed a novel Device Thrombogenicity Emulator (DTE) methodology that interfaces numerical and experimental approaches to optimize the thrombogenic performance of the device and reduce the bleeding risk associated with anticoagulation therapy. Device Thrombogenicity Emulator uses stress-loading waveforms in pertinent platelet flow trajectories that are extracted from highly resolved numerical simulations and emulates these flow conditions in a programmable hemodynamic shearing device (HSD) by which platelet activity is measured. We have previously compared two MHV, ATS and the St. Jude Medical, and demonstrated that owing to its nonrecessed hinge design, the ATS valve offers improved thrombogenic performance. In this study, we further optimize the ATS valve thrombogenic performance, by modifying various design features of the valve, intended to achieve reduced thrombogenicity: 1) optimizing the leaflet-housing gap clearance; 2) increasing the effective maximum opening angle of the valve; and 3) introducing a streamlined channel between the leaflet stops of the valve that increases the effective flow area. We have demonstrated that the DTE optimization methodology can be used as test bed for developing devices with significantly improved thombogenic performance.