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Patients who receive prosthetic heart valve (PHV) implants require mandatory anticoagulation medication after implantation due to the thrombogenic potential of the valve. Optimization of PHV designs may facilitate reduction of flow-induced thrombogenicity and reduce or eliminate the need for post-implant anticoagulants. We present a methodology entitled Device Thrombogenicty Emulator (DTE) for optimizing the thrombo-resistance performance of PHV by combining numerical and experimental approaches. Two bileaflet mechanical heart valves (MHV) designs, St. Jude Medical (SJM) and ATS, were investigated by studying the effect of distinct flow phases on platelet activation. Transient turbulent and direct numerical simulations (DNS) were conducted, and stress loading histories experienced by the platelets were calculated along flow trajectories. The numerical simulations indicated distinct design dependent differences between the two valves. The stress loading waveforms extracted from the numerical simulations were programmed into a hemodynamic shearing device (HSD), emulating the flow conditions past the valves in distinct 'hot-spot' flow regions that are implicated in MHV thrombogenicity. The resultant platelet activity was measured with a modified prothrombinase assay, and was found to be significantly higher in the SJM valve, mostly during the regurgitation phase. The experimental results were in excellent agreement with the calculated platelet activation potential. This establishes the utility of the DTE methodology for serving as a test bed for evaluating design modifications for achieving better thrombogenic performance for such devices., (2010 Elsevier Ltd. All rights reserved.)

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.

The development of actively hemostatic wound dressings for use in severe trauma remains a major public-health and military goal. But, although some manufacturers claim that existing dressings activate platelets and/or blood coagulation, mechanistic evidence is often lacking. We describe a method for assessing the active hemostatic properties of dressings in vitro, entailing measurement of the flow of recalcified platelet-rich plasma (PRP) through a dressing sample. If the dressing is hemostatically active, flow is reduced. This flow is then compared with the flow-through of PRP in which both platelet and coagulation function are blocked with EDTA. The ratio of the two generates a hemostatic index that ranges from 1.0 (no active hemostasis) to 0 (highly potent). The method is applicable to porous or semiporous dressings, whether fabric, sponge, fleece, or granules. For an active dressing, the test is easily modified to differentiate between the contributions of platelet and coagulation to overall hemostasis. The method is illustrated for fabrics, over-the-counter gauze and sponge dressings, collagen-based sheets, and an absorbent granule dressing. One active collagen dressing is used to illustrate discrimination between platelet and coagulation function. The ability to assess hemostatic properties may significantly enhance the development of advanced active dressings., ((c) 2008 Wiley Periodicals, Inc.)

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.

TiO(2) nanostructures with fascinating morphologies like cubes, spheres, and rods were synthesized by a simple microwave irradiation technique. Tuning of different morphologies was achieved by changing the pH and the nature of the medium or the precipitating agent. As-synthesized titania nanostructures were characterized by X-ray diffraction (XRD), UV-visible spectroscopy, infrared spectroscopy (IR), BET surface area, photoluminescence (PL), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques. Photocatalytic dye degradation studies were conducted using methylene blue under ultraviolet light irradiation. Dye degradation ability for nanocubes was found to be superior to the spheres and the rods and can be attributed to the observed high surface area of nanocubes. As-synthesized titania nanostructures have shown higher photocatalytic activity than the commercial photocatalyst Degussa P25 TiO(2).

Cigarette smoke has been shown to increase platelet activation and endothelial cell (EC) adhesion molecule expression. In the present study, we utilized a hemodynamic shearing device (HSD) to investigate the above effects in vitro in a combined system of platelets and cultured HUVECs (Human Umblical Vein ECs) under physiological shear stress. We investigated the alteration of E-selectin expression on ECs upon exposure to: (1) platelets and nicotine-free smoke extract (NFE), (2) platelets alone, (3) NFE alone, under physiological shear stress. We additionally confirmed the protective effect of nicotine on platelet activation. We found that: (i) surface expression of E-selectin on ECs was significantly increased upon simultaneous exposure of ECs and platelets to NFE relative to exposure of ECs to either platelets or NFE alone (p < 0.05). (ii) Platelet activation was significantly increased in the presence of NFE (p < 0.05). (iii) Nicotine (200 nM) when added to NFE, significantly reduced platelet activation due to NFE (p < 0.05), an effect additionally confirmed by conventional cigarette extracts which contain nicotine (p < 0.05). We therefore conclude that: (a) NFE and platelets additively increase EC E-selectin surface expression, and (b) nicotine modulates platelet activation regardless of ECs.

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.

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.

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.

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.

Polymer heart valves have been under investigation since the 1960s, but their success has been hampered by an overall lack of durability mainly due to calcification of the leaflets and a relatively high rate of thromboembolic complications. A new polymer (Quatromer) trileaflet design was tested for its thrombogenic potential and was compared to that of existing prosthetic heart valves routinely implanted in patients: a St. Jude Medical bileaflet mechanical heart valve (MHV) and a St. Jude porcine bioprosthetic tissue valve. The valves were mounted in a left ventricular assist device and the procoagulant activity of the platelets was measured using a platelet activation state (PAS) assay. The PAS measurements indicated that the platelet activation level induced by the polymeric valve was very similar to that induced by the St. Jude Medical MHV and the St. Jude tissue valve. No significant difference was observed between the three valves, indicating that they have a comparable thrombogenic potential.

Mathematical analysis of positive feedback loops in proteolytic systems has previously suggested that when the active enzymes are subject to inhibitory control these systems will exhibit threshold behavior. This is demonstrated in the present study, for the autolytic activation of factor XII in the presence of a contact activator and an irreversible inhibitor of factor XIIa. The threshold between the two system states - complete factor XII activation, or complete stability - is dependent on the kinetic balance between the catalytic rate of autoactivation and rate of enzyme (factor XIIa) inhibition. Activation of the system can be brought about by either increasing the catalytic rate (in this study, by using more potent contact-activation conditions), or by lowering the enzyme inhibition rate. Previous mathematical work predicted complete stability in a positive-feedback system that is below threshold, and this has been experimentally confirmed.

It is widely accepted that shear stress activates platelets. However, this may have two linked but separate causes: a direct effect of shear stress on individual platelets, and secondary inter-platelet activation dependent on the release of agonists caused by shear. Gel-filtered platelets were exposed to intermittent low shear at 20,000 and 200,000 platelets/microl and their activation was measured with a prothrombinase-based assay. At the lower count, activation was slow and essentially linear, but at the higher count, it rose exponentially with time, leading to 3-fold more prothrombinase activity. Inclusion of apyrase and/or prostaglandin I(2) slightly reduced activation at high platelet counts, but did not abolish the nonlinear kinetics, and antibodies against von Willebrand factor had no significant effect. The contributions of anionic phospholipid and factor Va to the prothrombinase activity were assessed by measurements in the presence of exogenous factor Va. The results strongly suggest that anionic phospholipid appearance is caused directly by shear exposure, but that factor Va release from the alpha-granules is a secondary event and largely the result of platelet-platelet signaling.

Mainstream and sidestream smoke extracts of both high-tar and low-tar cigarettes have been shown to increase platelet activation directly and to sensitize them to further activation by exposure to mechanical stimuli such as shear stress. However, nicotine has an inhibitory effect on platelet activation, opposite to that of whole cigarette smoke extracts. To distinguish between the nicotine-dependent and non-nicotine-dependent effects of smoke, platelets were exposed to mainstream and sidestream smoke extracts of low-nicotine and zero-nicotine cigarettes in vitro under flow conditions comparable with the normal circulation, and their activation state was measured using a modified prothrombinase-based assay. Both low-nicotine and zero-nicotine extracts caused increased platelet activation upon exposure to shear stress, and they caused significantly greater activation than extracts from ordinary high-tar, high-nicotine cigarettes. That nicotine was crucial was confirmed by adding 50 nM nicotine (comparable with the nicotine level in smokers' plasma) to zero-nicotine smoke extracts and demonstrating a reduction in the shear-dependent rate of platelet activation of more than 75%.

A study was conducted to measure in vitro the procoagulant properties of platelets induced by flow through Carbomedics bileaflet and Bjork-Shiley monoleaflet mechanical heart valves (MHVs). Valves were mounted in a left ventricular assist device, and platelets were circulated through them under pulsatile flow. Platelet activation states (PAS) were measured during circulation using a modified prothrombinase method. Computational fluid dynamics (CFD) simulations of turbulent, transient, and non-Newtonian blood flow patterns generated by the two valve designs were done using the Wilcox k - w turbulence model, and platelet shear-stress histories (the integral of shear-stress exposure with respect to time) through the two MHVs were calculated. PAS measurements indicated that the bileaflet MHV activated platelets at a rate more than twice that observed with the monoleaflet MHV. Turbulent flow patterns were evident in CFD simulations for both valves, and corroborated the PAS observations, showing that, for particles close to the leaflet(s), shear-stress exposure in the bileaflet MHV can be more than four times that in the monoleaflet valve.

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.

Circulating blood platelets regulate the initial phase of the hemostatic response through adhesive and aggregatory events and by providing the necessary procoagulant surface for prothrombinase complex assembly and thrombin generation. The signaling pathway(s) that regulate platelet procoagulant activity are largely unknown, although they are distinct from platelet aggregatory signals linked to fibrinogen ligation to the conformationally active alpha(IIB)beta(3) integrin. We describe a novel intracellular signaling mechanism involving platelet IQGAP1 that specifically regulates the development of platelet procoagulant activity under conditions of mechanical shear stress. Murine platelets that are deficient in IQGAP1 demonstrate increased prothrombinase activity compared with wild-type littermate controls when activated by a physiological shear stress of 16 dynes/cm(2) (shear rates of 1600 s(-1)) (p < 0.0001), corresponding to approximately 2.5 times the normal shear stress, or approximately 40% degree of stenosis in coronary arteries. The exaggerated prothrombinase activity is not associated with enhanced platelet microvesiculation (cytoskeletal proteolysis) and occurs independently of the intracellular calcium release, [Ca(2+)](i), but it is specifically coupled to the alpha-granule exocytic pathway without concomitant effects on aminophospholipid exposure. These observations identify platelet IQGAP1 as an important modulator of normal hemostasis and as an appropriate pharmacological target for control of platelet procoagulant function.

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.

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.

Measurement of small changes in platelet activation state (PAS) in circulating stenotic systems in vitro has been problematic because of a paucity of real-time assay methods and circulation systems of low platelet-activating potential. PAS was measured by a modified prothrombinase assay in which activated platelets provide the essential cofactors in the activation of prothrombin by factor Xa. Chemical modification of the prothrombin ensures that the thrombin produced, while assayable, does not activate platelets. Human platelets were circulated in loops in which exposure to shear stress was adjusted by independently varying flow rate, viscosity, and the time of exposure to shear. Although with some differences in platelet response to different conditions of stress, the PAS directly increased with time of circulation, shear stress, and time of exposure to shear. The results show that low-level platelet activation caused by shear stress in a circulation loop can be quantitatively assessed in near-real time in a system of tube geometry. They confirm previous results obtained in non-circulating systems that exposure of platelets to shear conditions on the same order as found in the vasculature causes significant platelet activation, and that this activation is dependent on both shear stress and time of exposure.

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.

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.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state., (Copyright 1999 Academic Press.)

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus displaying several attractive features applicable to haemophilia A gene therapy, including nonpathogenicity and potential for long-term transgene expression from either integrated or episomal forms. We have generated and characterized two B-domain-deleted (BDD) fVIII mutants, deleted in residues Phe756 to Ile1679 (fVIIIdelta756-1679) or Thr761 to Asn1639 (fVIIIdelta761-1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD-fVIII of the predicted size in both lysates and supernatants (Mr approximately 155 kD for fVIIIdelta756-1679 and Mr approximately 160 kD for fVIIIdelta761-1639) after transient transfection into COS-1 cells. Functional fVIII quantification appeared maximal using fVIIIdelta761-1639, as evaluated by Coatest and clotting assay (98+/-20mU/ml/1x10(6) cells and 118+/-29 mU/ml/1x10(6) respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIdelta761-1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1-1) promoter, and demonstrated VIII activity approximately 30% of that seen using CMV promoter. This BDD-fVIII (rAAV(pHU1-1) fVIIIdelta761-1639) can be efficiently encapsidated into rAAV (107% of wild type), as demonstrated by replication centre and DNAase sensitivity assays. A concentrated recombinant viral stock resulted in readily detectable factor VIII expression in COS-1 cells using a maximally-achievable MOI approximately 35 (Coatest 15 mU/ml; clotting assay 25+/-20 mU/ml/1x10(6) cells). These data provide the first evidence that rAAV is an adaptable virus for fVIII delivery, and given the recent progress using this virus for factor IX delivery in vivo, provide a new approach towards definitive treatment of haemophilia A.

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF-pre-treated cells with prothrombin and factors X, Va, and VIIa at 37 degrees C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti-thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum-free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP-2 abrogated thrombin-induced progelatinase A activation, it had no significant effect on thrombin-induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF-induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane.

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.

The inhibition by tissue factor pathway inhibitor (TFPI) of its two target enzymes--factor Xa and the tissue factor-factor VIIa complex (TF:VIIa)--has been studied under near-physiological reactant concentrations and conditions. Over a TFPI range of 0-1 nM, the rate of inhibition of factor Xa, in the presence of Ca2+ and anionic phospholipid vesicles at 37 degrees C, was proportional to TFPI concentration, giving an association rate, k1, of 0.96 x 10(9) M-1 min-1. Factor Xa inhibition did not proceed to completion, the reaction attaining a near-equilibrium that was dependent on the TFPI concentration. The estimated dissociation rate of the TFPI:Xa complex, k-1, was independent of TFPI concentration, with a mean value of 0.02 min-1. The resulting calculated value of K1, the apparent dissociation constant for the initial step, is 21 pM. Slow decay of the remaining factor Xa in such incubations, detectable after attainment of the rapid initial near-equilibrium, confirmed the two-step mechanism proposed by Huang et al. (1993) [J. Biol. Chem. 268, 26950-26955], but did not permit determination of a rate constant for the second step. Omission of anionic phospholipid had no significant effect on either k1 or k-1. A high-molecular-weight fraction of heparin, at saturating levels (> or = 0.05 unit/mL, congruent to 25 nM), increased k1 2-fold, with no detectable effect on k-1. The second stage of TFPI action was studied by preformation of the TFPI:Xa complex, and its incubation with the TF:VIIa complex in the presence of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)

A model of a proteolytic positive-feedback loop, similar in general terms to feedback loops that occur in blood coagulation and other systems, has been examined by both explicit and numerical analysis. In this loop, modeled as a closed system, each enzyme (E1, E2) catalyzes the formation of the other from its respective zymogen (Z1, Z2), and both enzymes are subject to irreversible inhibition. The system shows three major characteristics. (1) No significant Z1 or Z2 activation occurs unless the combination of initial conditions and kinetic parameters is above a threshold level. This threshold occurs when the product of the enzyme generation rates equals the product of their inhibition rates. When the formation-rate product is less than the inhibition-rate product, there is no response: E1 and E2 generation is minimal and the lag time is effectively infinite. Conversely, when the generation-rate product exceeds the inhibition-rate product, explosive formation of both E1 and E2 is seen. For responses exceeding the threshold, the following obtain. (2) The lag time in E1 and E2 generation is a highly nonlinear function of the zymogen concentrations and the enzyme generation and inhibition rates. In contrast, there is a simple logarithmic relationship between the lag time and the initial trace concentration of the enzyme that is responsible for initiating the system; in this model, E1. (3) The extent of Z1 and Z2 activation is similarly a nonlinear function of the conditions and parameters but is independent of the initiating trace level of E1.(ABSTRACT TRUNCATED AT 250 WORDS)

The decay of human coagulation factor VIIIa has been studied by kinetic methods that ensure no interference through proteolytic feedback. The rate of decay of factor VIIIa activity was found to vary with the activator used to activate factor VIII. Thrombin-activated factor VIII-von Willebrand factor complex (fVIII-vWf) decayed at a rate of 0.31 min-1, whereas factor Xa-activated fVIII-vWf decayed at 0.11 min-1 under the same conditions. Factor VIII free of von Willebrand factor (factor VIII: C), although decaying at a generally slower rate after activation, still showed a dependence of decay rate on activator: thrombin-activated factor VIII:C decaying at a rate of 0.06 min-1, and factor Xa-activated factor VIII: C at 0.01 min-1. Readdition of von Willebrand factor (18 micrograms/ml) to factor VIII:C did not alter the observed activity or decay rate. The decay of the two species of factor VIIIa was studied, using the fVIIIa-vWf complex, in the presence of varying levels of factor IXa. Plots of reciprocal decay rates vs factor IXa concentration were linear, and nearly parallel for the two factor VIIIa species, with a mean slope of 0.56 min.nM-1. In addition to these studies, we have confirmed previous studies showing that the two forms of factor VIIIa differ in cofactor activity, but they do so in the same ratio as in their decay rates. We suggest that this difference and that observed in decay rate have a common cause, and incorporate this into a potential kinetic model of factor VIII activation and decay.

A simple numerical model of the activation of factor X by factors IXa and VIII has been constructed in order to identify and examine the major controls that operate in a nonflowing system in the presence of (1) inhibitors of factor Xa and (2) feedback activation of factor VIII by factor Xa. The model confirms, and allows parameter estimation for, (1) the control of factor Xa yield by factor VIIIa decay; (2) the control of generation-curve area by the rate of factor Xa inhibition; and (3) the reduction in the factor VIIIa decay rate in the presence of factor IXa. Beyond confirmation of existing data, the model also predicts that below a definite, but very low, threshold level of factor IXa (less than or equal to 10 pM), minimal feedback activation of factor VIII will occur. The concentration of factor IXa at which the threshold is observed in simulations is dependent on the rate of inhibition of factor Xa.

The modification of human factor X by 2-sulfo-N-succinimidyl acetate was investigated and shown to produce a factor X species which, when activated, has no activity toward factor VIII. Acylation of factor X (0.9 microM) was carried out in the presence of 1 mM calcium at different reagent concentrations and pH values at 22 degrees C for time courses up to 1 h. Optimal modification was achieved using 0.3 mM reagent at pH 8.0 for 30 min. The modified zymogen, acetylated factor X, is activated at full rates by factor IXa/VIIIa and by the factor X-activating protein of Russell's viper venom. The activated product, acetylated Xa, has an enhanced amidolytic activity (110%) but has almost no detectable clotting activity (0.1%). More importantly, we have shown that acetylated Xa, in contrast to native Xa, does not activate factor VIII. This allows accurate quantitation of factor VIII activation without complications due to positive feedback reactions. We have demonstrated this in an examination of the activation of factor VIII by factor IXa.

Commonly used manual and calculator methods for analysis of clinically important parallel-line bioassays are subject to operator bias and provide neither confidence limits for the results nor any indication of their validity. To remedy this, the authors have written a general program for statistical analysis of these bioassays for the IBM Personal Computer and its compatibles. The program has been used for analysis of bioassays for specific coagulation factors and inflammatory lymphokines and for radioimmunoassays for prostaglandins. The program offers a choice of no transform, logarithmic, or logit transformation of data, which are fitted to parallel lines for standard and unknown. It analyzes the fit for parallelism and linearity with an F test, and calculates the best estimate of the result and its 95% confidence limits. Comparison of results calculated by PARLIN with those previously obtained manually shows excellent correlation (r greater than 0.99). Results obtained using PARLIN are quickly available with current assay technics and provide a complete evaluation of the bioassay at no increase in cost.

An enzyme-linked immunoabsorbent assay (ELISA) has been developed for the measurement of the complex of human antithrombin and Factor Xa. Rabbit anti-human Factor X antibodies are adsorbed to ELISA plates, and samples containing Xa-antithrombin complex are added. This is followed by the addition of F(ab')2 fragments of rabbit antibodies against human antithrombin, previously labeled with alkaline phosphatase, and subsequent measurement of the bound labeled antibody by hydrolysis of p-nitrophenylphosphate. The minimum level of complex detectable in a sample is ca. 0.1 nM. The assay has been used to follow the generation of Xa-antithrombin complex in kinetic situations by the addition of 1 microM Ile-Glu-Gly-Arg-chloro- methylketone to the ELISA sampling buffer, and it has also been used in plasma systems, where a 20-fold reduction in the sensitivity of the assay is observed. This reduction was shown to be entirely caused by the plasma Factor X. The assay has been used to follow generation of the Xa-antithrombin complex in defibrinated plasma upon activation of the clotting system with the Factor X-activating protein of Russell's Viper venom, and has been compared with the total generation of Factor Xa, measured by a radiopeptide assay of Factor X activation in the same mixtures.

The activation of Factor IX by tissue factor-Factor VII has been studied in a bovine plasma system under conditions that minimize the activation of Factor VII. The plasma was defibrinated, then passed twice through a column of anti-Factor X coupled to Sepharose in order to lower the Factor X level below its limit of assay (ca. 5 ng/ml), and once through an anti-Factor IX column to remove Factor IX. Varying levels of tritium-labelled Factor IX were then added back to the plasma, permitting measurement of its activation upon the addition of tissue factor and Ca2+. Despite the absence of significant levels of Factor X in the system, the course of Factor IX activation was initially characterized by some upward curvature, which suggested activation of the plasma Factor VII during the incubation. In order to obtain linear activation of Factor IX three proteolytic inhibitors were added to the system: 1) a Factor Xa inhibitor, 1,2-bis-(5-amidinobenzimidazole)-ethane, 2) aprotinin, and 3) heparin. Under these conditions the apparent Km of non-activated Factor VII (+ tissue factor) on Factor IX was 17.3 +/- 2.5 nM (SE), and the maximum velocity was 0.12 nM/min. In parallel experiments the plasma Factor VII was activated by first treating the plasma with Factor Xa for 30 seconds before the addition of inhibitors and the final addition of substrate. Under these conditions the maximum velocity rose to 4.2 nM/min, and the Km increased to 53.3 +/- 6.0 nM (SE). This change in the Km is highly significant (P less than 0.002), and indicates that the activation of Factor IX by nonactivated plasma Factor VII cannot be due only to traces of Factor VIIa in the plasma. At least in part, activation of Factor IX in the presence of tissue factor is suggested to be a result of the action of Factor VII itself.

The basis of the specificity of human coagulation factor Xa has been probed with a reagent that reacts with nucleophiles, N-succinimidylpropionate. At pH 8.0 and 0.25 mM N-succinimidylpropionate, 0.4 microM factor Xa lost approx. 90% of its activity toward prothrombin in 4 min. The decay was first-order, k = 0.64 min-1, which increased to 0.98 min-1 in 1 mM Ca2+, and the dependence of k upon pH was consistent with primary amines being the target. The rate of modification was unaffected by the presence of a tetrapeptide substrate during modification; likewise, activity toward a tripeptide p-nitroanilide was unaltered during exposure of factor Xa to N-succinimidylpropionate with or without Ca2+. In addition, inhibition by antithrombin III was retained with a somewhat enhanced rate after modification; however, the acceleration of this by heparin was significantly less. Kinetic determination of the number of residues modified gave a reaction order of 2.0, while reaction with N-succinimidyl[3H]propionate yielded labeled factor Xa containing 1.0 mol N-succinimidylpropionate/mol factor Xa and 50% normal clotting activity, or 2.0 mol N-succinimidylpropionate/mol and 1% activity, respectively. Thus, one nucleophilic group is required for the reaction of factor Xa with prothrombin but not for the hydrolysis of peptides or recognition of antithrombin III. The decay of clotting activity of the factor X zymogen in N-succinimidylpropionate was much slower though still Ca2+-dependent. Conversely, the reaction of a related compound--N-succinimidyl(4-hydroxyphenyl)propionate or Bolton-Hunter reagent--with factor Xa broadly resembled that of N-succinimidylpropionate but the decay curves indicated more complex kinetics. Therefore, the target groups vary in their accessibility to modification according to the structural characteristics of both the protein and the reagent.

A method has been developed for detailed kinetic studies of the inhibition of factor Xa in human plasma. Radiolabeled enzyme is not required, and the method can be used at initial factor Xa levels of 1 nM. The method is discontinuous and based on the removal of samples into an amidolytic assay done in the presence of 1% Lubrol-PX detergent. This permits the study of inhibition in mixtures containing phospholipid, platelets, or thromboplastin. The method can be used at inhibition rates in excess of 1 min-1, and by suitable analysis can be used to estimate the contribution of inhibition by alpha 2-macroglobulin, which does not itself inhibit amidolytic activity. The method is at present limited to cases where thrombin is not generated in large excess. Factor Xa inhibition has been studied in citrated plasma as a function of total plasma concentration, and--by the use of antithrombin-depleted plasma--as a function of the antithrombin concentration of the plasma. In all situations inhibition is characterized by second-order behavior: (i) total inhibition rate is proportional to plasma concentration up to 95%, giving a maximum rate in the absence of calcium of 1 min-1; (ii) inhibition in depleted plasma reconstituted with antithrombin shows inhibition rate to remain linearly related to antithrombin concentration; and (iii) the estimated rate due to alpha 2-macroglobulin is proportional to plasma concentration. It is thus confirmed that, as in pure systems, inhibition of factor Xa in whole plasma is linearly related to the concentration of each class of inhibitor.

Protein C and protein S are vitamin K-dependent coagulation factors that together act as an anticoagulant, and antithrombin III is a plasma protein that inhibits several activated factors in the coagulation cascade. Although deficiencies of any of these three proteins have been associated with deficiencies of these factors. We report one patient with a protein S deficiency, another with a protein C deficiency, and a third with an antithrombin III deficiency, each of whom who had extensive arterial thrombosis. We suggest that deficiencies of these proteins may constitute risk factors for arterial thrombosis.

The preparation of activated Factor X from reaction mixtures of bovine Factor X and Russell's-viper venom is described. The molecular weight of purified protein varies about a mean value of 40000; this variation is the result of at least two forms of Factor Xa. The action of activated Factor X, together with purified Factor V, was studied on purified prothrombin and the reaction products were isolated. In addition to thrombin, two other polypeptides with molecular weights of 16000 and 19500 were recovered.

1. A method is described for the simultaneous isolation of both Factor X and prothrombin from bovine plasma. The proteins are adsorbed on and eluted from barium sulphate and chromatographed on DEAE-Sephadex and are finally purified by rechromatography on DEAE-Sephadex. 2. The proteins can be purified in 48h from the collection of the blood and the method can be used to process large volumes of plasma. 3. The prothrombin has a molecular weight of 70300; the Factor X, on the other hand, is polydisperse, with most of the protein (86%) having a molecular weight of 56000.