Notre équipe s’intéresse à la relation structure-fonction d’une protéine, l’antithrombine (AT), un inhibiteur physiologique de la coagulation, en vue d’un développement thérapeutique. Cette protéine anticoagulante, capable de lier un motif pentasaccharidique sur les dérivés hépariniques, possède en outre, à fortes concentrations (500%), des propriétés anti-inflammatoires médiées par sa liaison aux héparan-sulfates cellulaires. Ce profil a mené à l’évaluation de l’AT dans des situations associant un emballement de la coagulation et de l’inflammation, comme c’est le cas au cours du sepsis sévère et d’autres situations d’ischémie-reperfusion (I/R). Cependant, les fortes concentrations utilisées dans les études précliniques nécessiteraient d’administrer des doses d’AT incompatibles avec le profil de sécurité de cette protéine anticoagulante.Dans ce contexte, nous avons, au cours de ce travail, caractérisé un variant d’AT (AT-N135Q-Pro394) dépourvu d’activité anticoagulante et doué d’une affinité augmentée pour l’héparine. Ce variant est capable de piéger des dérivés hépariniques et apparait comme un candidat idéal pour une utilisation comme antidote en cas de surdosage en héparine non fractionnée (HNF), héparines de bas poids moléculaire (HBPM) ou fondaparinux. Par ailleurs, ce variant pourrait être utilisé à des doses cytoprotectrices, sans risque hémorragique.Afin de tester cette dernière hypothèse, nous avons développé un modèle d’I/R rénale chez la souris, qui s’accompagne d’une augmentation significative de marqueurs de dysfonction rénale (Urée, Créatinine, Kim-1) et de l’inflammation (expression tissulaire de cxcl-1, il-6). L’AT avait déjà été montrée protectrice (Mizutani et al, Blood 2003) dans un modèle murin comparable. De façon surprenante, nous n’avons observé aucun des effets protecteurs décrits, ni sur l’inflammation ni sur la fonction rénale, et que ce soit avec de l’AT plasmatique, de l’AT recombinante ou encore avec un mélange équimolaire d’AT latente et native. Ce même modèle nous a pourtant permis de mettre en évidence les effets nephroprotecteurs et anti-inflammatoires d’une autre protéine anticoagulante, la protéine C activée. Ces résultats décevants font écho à la rétractation pour fraude de l’article de Mizutani et al. en 2013. Le travail approfondi que nous avons mené nous permet de clarifier la littérature et d’affirmer que l’AT, d’origine plasmatique ou recombinante, ne possèdent pas d’effet protecteur dans l’I/R rénale chez la souris. Dans ces conditions, le variant AT-N135Q-Pro394 n’a pas été testé.Concernant la seconde indication, l’AT-N135Q-Pro394 avait déjà été évaluée in vivo comme antidote aux dérivés hépariniques, HNF, HBPM et fondaparinux, avec d’excellents résultats. Néanmoins, cet effet antidote a été exploré spécifiquement par mesure de l’activité anti-facteur Xa alors que l’AT inhibe plusieurs enzymes de la cascade de coagulation tel que les facteurs VIIa, IXa et IIa. Nous avons donc exploré cet effet antidote dans un test plus global de la coagulation, le test de génération de thrombine (TGT) pour pouvoir le comparer aux autres stratégies non spécifiques utilisées pour antagoniser les dérivés hépariniques (facteur VII activé recombinant, concentré de complexes prothrombiques activés ou protamine). De façon intéressante, dans un plasma mimant un surdosage, notre variant présente un effet antidote supérieur aux agents hémostatiques et au sulfate de protamine vis-à-vis du fondaparinux et des HBPMs, respectivement, et équivalent au sulfate de protamine vis-à-vis de l’HNF. Enfin, dans du plasma en l’absence d’anticoagulant, l’AT-N135Q-Pro394 ne montre aucun effet sur la génération de thrombine contrairement aux agents hémostatiques et au sulfate de protamine qui, ajoutés seuls dans du plasma, modifient significativement le profil des TGT / Our team topic focuses on the structural-function relationship of a natural anticoagulant, antithrombin (AT), in order to develop potential therapeutic agents. AT inhibits several serine proteases of the coagulation cascade and its inhibitory activity is increased when AT binds to a pentasaccharidic motif contained within in the heparin derivatives. At high concentrations (500%), AT also exerts anti-inflammatory and cytoprotective properties through its binding to heparan sulfate proteoglycans, making it a good candidate for supportive therapy in clinical settings associating inflammation and coagulation activation. Indeed, AT has already been evaluated in vivo in various models of ischemia-reperfusion injury (IRI) and AT even reached a large-scale clinical trial in severe sepsis. However, the high concentrations of AT that are needed to exert anti-inflammatory properties are inconsistent with the safety profile of this anticoagulant protein.In this context we have further characterized an AT variant (AT-N135Q-Pro394) with increased affinity to heparin but devoid of anticoagulant activity. Indeed, this variant was described to be able to trap heparin derivatives and our work was to pursue the characterization of this variant as an antidote toward heparin derivatives in clinical situations of overdosing. In addition, this AT variant binds to heparan sulfate proteoglycans with higher affinity, as compared to native AT, and appears as a promising cytoprotective agent whose administration would not be associated with any bleeding risk.To test the latter hypothesis, we developed a murine model of renal IRI in which the renal function was severely impaired, as attested by increased kidney injury markers (urea, creatinine, kim-1) and local kidney inflammation (renal gene expression of il-6 and cxcl-1). Indeed, in 2003, Mizutani et al. reported a protective effect of AT in a similar murine model of renal IRI. Surprisingly, we observed none of the described protective effects, neither on inflammation nor renal function, with plasma AT, recombinant AT and an equimolar mixture of native and latent AT. Nevertheless, the same model enabled us to highlight the nephroprotective and anti-inflammatory properties of another anticoagulant protein, activated protein C (APC), as previously reported. These disappointing results coincided with the withdrawal in 2013 of the study of Mizutani et al., and our work allowed us to clarify the literature and to claim that neither recombinant nor plasma-derived native nor latent forms of AT exhibit a protective effect in renal IRI in mice. Under these conditions, AT-N135Q-Pro394 variant has not been tested in our model.AT-N135Q-Pro394 has also been previously shown to efficiently neutralise the anticoagulant activity of heparin derivatives, including unfractionated heparin (UFH), low molecular weight heparins (LMWH) and fondaparinux in vivo. Nevertheless, this reversal effect was only explored by anti-factor Xa assays whereas AT inhibits a number of coagulation proteases, including factors VIIa, IXa and IIa. Therefore, we explored AT-N135Q-Pro394 variant in a more global coagulation assay, the thrombin-generation assay (TGA), in order to compare its activity with non-specific reversal agents used toward heparin derivative overdose (recombinant-activated factor VII, activated prothrombin-complex concentrate or protamine). Interestingly, in plasma mimicking an overdose, our variant demonstrated greater reversal efficiency as compared to hemostatic agents and protamine sulfate toward fondaparinux and LMWH, respectively, and was as efficient as protamine sulfate toward UFH. Finally, when added to native plasma (in the absence of heparin derivative), AT-N135Q-Pro394 showed no effect on thrombin generation unlike hemostatics and protamine sulfate that all significantly affect the TGA profile

Cette recherche s’inscrit dans un contexte d’amélioration des performances et du confort des vêtements textiles d’intervention contre le feu et la chaleur. L’objectif est de réduire le poids total des équipements de protection individuelle (EPI), en remplaçant leur structure actuelle par un tissu complexe de type « Spacer Fabric », qui résulte de l’assemblage de deux couches, aux propriétés différentes, par un fil de liage. Ces travaux de thèse se concentrent sur le développement du fil de liage et peuvent se définir comme une étude de faisabilité visant à prouver la possibilité de filer des monofilaments de « gros » diamètre (200 – 300 μm) à partir d’une solution de polyamide-imide Kermel®. Pour mener ce projet à terme, un dispositif expérimental de filage par coagulation a été développé. Entièrement automatisé, il permet aussi bien l’étude de l’influence des paramètres de filage sur la qualité des monofilaments obtenus, que la simulation d’une production de type industriel. Pour répondre à notre problématique, cet outil de prototypage a été appliqué au développement des monofilaments souhaités. Un plan d’expériences a été construit, comprenant quatre facteurs : le diamètre d’extrusion, la concentration en solvant du bain de coagulation et les taux d’étirage appliqués dans le bain et dans l’air. Cette étude expérimentale a mis en évidence l’importance de la maîtrise de la cinétique de coagulation, avec une influence hautement significative des facteurs diamètre d’extrusion et concentration du bain de coagulation, notamment sur l’évolution du taux de vide des monofilaments obtenus. / This research aims to improve the performance and the comfort requirements of textile protective clothing against heat and flames. The main purpose is to reduce the weight of such personal equipment by replacing their current structure with complex woven “Spacer Fabric”, composed of an assembly of two textile layers, having different properties, and linked by a binder yarn. These works particularly focus on the development of this binder yarn and consist in a feasibility study to demonstrate the possibility to demonstrate the possibility to spun monofilaments having a large diameter (200 – 300 μm) from a polyamide-imide solution (Kermel®). A dedicated experimental wet-spinning device has been designed to carry out this project. Such fully automated machine enables to conduct detailed studies concerning the influence of multiple spinning parameters on the physical properties of the manufactured monofilaments, therefore providing a simulation of an industrial production of such fibers. Hence, this prototyping tool has been used to develop monofilaments addressing the initially stated issue. A four factor Design of Experiments has been built to investigate the effects and combinations between the following parameters: extrusion diameter, concentration of the coagulation bath, drawing rates applied in the bath and in the air. The derived results mainly showed the importance of controlling the coagulation kinetic. Moreover, highly significant influences of both extrusion diameter and concentration of coagulation bath factors have been highlighted, especially concerning the evolution of the void fraction of the monofilaments.

An investigation of the temperature response and corresponding lesion growth resulting from in vivo interstitial laser photocoagulation was performed in order to test the applicability of Arrhenius theory. The irradiations were performed in vivo in rabbit muscle for various exposures at 1.0W using an 805 nm diode laser source coupled to an optical fibre with a pre-charred tip, thereby forcing it to function as a point heat source. Temperature responses were measured using a five-microthermocouple array along a range of radial distances from the point heat source. Each temperature profile was fitted with a curve predicted by the Weinbaum-Jiji bioheat transfer equation. The lesions were resected 48 hours after irradiation and the boundary of thermal damage resulting in necrosis was determined histologically. Numerical integration of the Arrhenius integral using temperature-time data at the lesion boundary produced corresponding activation energy and pre-exponential factor pairs (Ea , a) consistent with reported values for various other endpoints and tissue types. As well, theoretical predictions of the lesion growth from Arrhenius theory agreed well with experimental results. However, the thermal parameters, which are generally assumed to be constant when solving the bioheat transfer equation, were found to vary with radial distance from the source, presumably due to a dependence on temperature. / Thesis / Master of Science (MS)

The objective of this preliminary study was to determine the potential of diffuse correlation spectroscopy (DCS) for assessment of coagulation depth. Coagulation of tissue can occur due to a number of different reasons such as thermal or electrical burns or radiofrequency ablation. DCS is a non-invasive optical technique which can be used to determine the optical and dynamic properties of tissue by fitting a theoretical model of photon propagation in multiply scattering tissue to experimental data obtained from measurements. The DCS measurements were performed on two-layered phantom models that represent healthy tissue with high flow properties with a layer of coagulated tissue with low flow properties on top. Three different phantom models were prepared using gelatin-Intralipid gels, PDMS, and nylon as an upper layer, and an Intralipid solution was used for the bottom layer for all three phantoms. DCS measurements were performed on all three phantom models with varying thicknesses of the upper layers, and varying source-detector separations. The acquired data from the DCS measurement were analyzed in MATLAB in order to obtain the electric field temporal autocorrelation function. A theoretical model describing photon propagation in a two-layered medium was fitted to the obtained data in order to extract the desired parameters. The results showed that the thickness of the gelatin-Intralipid gels could be extracted within a 0.5 mm certainty and the thickness of the PDMS phantoms could also be extracted within approximately 0.7 mm. For the nylon phantoms, the results obtained were not good because the fitting was not successful and the thickness was not extracted appropriately. There is potential in DCS for assessment of burn wound depth but further research and development has to be done in the field in order to obtain more accurate and reliable results.

Fluoride naturally occurs in some ground and surface waters at high concentrations all around the world. Due to increasing health concerns about over-exposure to fluoride in drinking water, the United States Environmental Protection Agency (USEPA) has begun to review fluoride as a drinking water contaminant. Should the USEPA decide to lower the fluoride maximum contaminant limit (MCL), many water systems in addition to those already struggling to meet the fluoride MCL will require defluoridation as part of their drinking water treatment process. Alum coagulation was investigated as a defluoridation treatment strategy in this research project. Surface and blended (ground/surface) drinking water sources with high fluoride concentrations pose a unique challenge to defluoridation by alum coagulation because of the presence of both natural organic matter (NOM) and fluoride. Defluoridation of synthetic and natural waters using jar tests elucidated interactions of fluoride, NOM, and aluminum during alum coagulation. Alum coagulation was able to remove 80% of fluoride from natural waters with a 500 mg/L alum dose; however, 50% fluoride removal was observed to be possible with an alum dose of 150-170 mg/L. The optimum pH for fluoride removal in synthetic and natural waters was observed to be approximately 6.5 and was found to be an important factor in determining the overall performance of alum coagulation. The presence of fluoride during alum coagulation was found to reduce the removal of three low molecular weight (LMW) organics, acting as surrogates for NOM, to different extents depending on their functionality. The presence of LMW organic acids in synthetic waters did not impact the removal of fluoride; however, increasing NOM concentrations in the natural waters likely accounted for decreasing fluoride removals observed in the natural waters. Additional jar tests with natural waters revealed that pH adjustment was unnecessary for defluoridation of high pH and high alkalinity waters and that an enhanced precipitation effect occurred at low alum doses when no pH adjustment was made during alum coagulation. The enhanced precipitation effect caused comparable or enhanced removals of fluoride and NOM to be observed despite system pH values being higher than the optimal defluoridation pH of 6.5. Lower aluminum residuals were also observed as part of the enhanced precipitation effect, suggesting that when precipitation begins under high pH conditions, fluoride interference does not occur and therefore promotes more precipitate formation with greater available surface area for adsorption. However, as precipitation occurs, pH drops, and fluoride increasingly interacts with the aluminum precipitate resulting in greater overall fluoride removals. / text

Historically, the presence of microalgae (algae hereafter) in natural waters has been viewed as a nuisance due to its adverse impact on water quality. More recently, however, algae are being investigated as potential sources of biofuel and a range of natural products. These applications require the development of large-scale cultivation systems for mass production that include growth, harvesting, concentration, and product recovery components. While challenges still remain with respect to many of the processes involved in mass production, one of the most technically and economically challenging steps is harvesting the algae from dilute growth cultures, especially in systems where chemical additives are of concern either within the algae concentrate or the effluent water. For this reason, a pH-induced flocculation/deflocculation method using the hydroxides of alkali or alkaline earth metals (e.g., lime, caustic soda) is of particular interest for algae harvesting as Na, Ca and Mg are typically present in natural waters. The goal of this research was to determine the underlying mechanisms responsible for algae coagulation by magnesium and calcium and to evaluate the potential of these mechanisms for harvesting algae for a range of synthetic and field source water chemistries. In the first two phases of this research, the mechanisms for coagulation with magnesium and calcium were studied independently. A series of bench-scale experiments were designed to isolate the potential mechanisms of algae destabilization associated with each of these cations as a function of water chemistry, and microscopic analyses were performed to characterize the flocculated algae/precipitate mixtures. In the third phase of this research, removal of algae in field source waters was evaluated with respect to the underlying science elucidated in the previous phases. The results indicate that the dominant algae destabilization mechanism associated with magnesium shifts from Mg adsorption/charge neutralization to Mg(OH)2(S) precipitation-enhanced coagulation with increasing pH. Moreover, dissolved Mg2+ adsorption to the algae surface led to effective algae coagulation, while minimizing the mass of precipitated Mg(OH)2(S). For Ca, this research identified the importance of the nucleation process (heterogeneous vs. homogeneous nucleation) on algae removal efficiency. Heterogeneous nucleation is a key factor for optimizing algae removal; thus, the degree of oversaturation with respect to CaCO3(S) is a crucial operating parameter. This research demonstrated that the algae harvesting process using pH-induced flocculation/deflocculation method can be optimized for a wide range of source waters if the water chemistry (e.g. pH, ion concentration, alkalinity, ionic strength) is properly incorporated into the system design. / text

<p>Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.</p><p> A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.</p><p> The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.</p><p> To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.</p> / Dissertation

Biomaterials are commonly used in the medical clinic today; however, artificial materials can activate the cascade systems in the blood (complement-, coagulation-, contact- and fibrinolytic systems) as well as the platelets to various degrees. When an artificial surface comes in contact with blood, plasma proteins will be adsorbed to the surface within seconds. The composition of the layer of proteins differs between materials and is crucial for the hemocompatibility of the material. This thesis includes five projects. In Paper I the anticoagulants heparin and the thrombin inhibitor hirudin were evaluated in a whole blood model. Hirudin was found to be superior to low dose heparin since it did not affect the activation of the complement system nor the leukocytes. The most interesting observation was that expression of TF was seen on surface-attached monocytes in hirudin- treated blood but not heparin blood. In Paper II peptides from the streptococcal M-protein, which has affinity for the human complement inhibitor C4BP, were attached to a polymeric surface. When being exposed to blood the endogenous complement regulator was enriched at the surface of the material, via the M-peptides. With this new approach we created a self-regulatory surface, showing significant lowered material-induced complement activation. In Paper III apyrase, an enzyme which hydrolyzes nucleoside ATP and ADP, was immobilized on a polymer surface. Lower platelet activation and platelet-induced coagulation activation was seen for the apyrase-coated surface compared to control surfaces after exposure to whole human blood, due to the enzymes capability to degrade ADP released from activated platelets. In Paper IV and V we synthesized an array of polymeric materials which were characterized regarding physical-chemical properties, adsorption of plasma proteins, and hemocompatibility. The polymers showed widely heterogeneous protein adsorption. Furthermore, when the polymers were exposed to whole blood, two of the materials showed superior hemocompatibility (monitored as complement- and coagulation activation), compared to the reference poly(vinyl chloride).

The majority of cancer related deaths occur due to the invasive growth of metastatic lesions. In the early stages of metastasis, circulating cell interact with the endothelial cells to establish at a distant site. In inflammation endothelial activation results in induction of adhesion molecules on the endothelium that participate in the homing of leukocytes. Because of the interactions of metastatic cells with the endothelium, the question was whether some of the characteristic molecules of endothelial activation were induced during metastasis. In vivo pulmonary metastatic models were used to characterize the expression profile of endothelial activation. Immunohistochemistry identified VCAM-1 to be induced on the pulmonary endothelium following tumour cell arrest. VCAM-1 upregulation was not observed prior to tumour cells arrest or within the first hours. In contrast, tumour cell arrest appeared to be required for endothelial activation, arguing against a mechanism analogous to leukocyte homing. The upregulation of VCAM-1 upon tumour cell arrest corresponded with the initiation of platelet clot formation around the tumour cell and recruitment of leukocytes to the site, both previously shown to be essential for metastasis. Disruption of both phenomena, either through genetic or pharmacological manipulation, demonstrated that in contrast to the recruited leukocytes, platelets were involved in inducing endothelial activation. Another protein investigated was VAP-1. In contrast to VCAM-1, central to VAP-1 adhesive function is its enzymatic activity. Blocking the functions of either molecule highlighted their role in facilitating the recruitment of the leukocyte population to the tumour cell. Disruption of which led to a significant attenuation of metastasis. While VCAM-1 and VAP-1 function appears critical in the early steps of metastasis, their inhibition had no effect at later stages of pulmonary colonization.

At sites of vascular injury, the critical blood clotting enzyme thrombin is generated from prothrombin via Prothrombinase, a macromolecular, Ca2+-dependent enzymatic complex consisting of the serine protease factor Xa and the non-enzymatic cofactor factor Va, assembled on the membranes of activated platelets. Platelets regulate thrombin formation by providing specific binding sites for the components of Prothrombinase and by releasing a platelet-derived factor V/Va molecule that is more procoagulant than its plasma counterpart and partially resistant to proteolytic inactivation. This dissertation identifies and characterizes the subpopulation of platelet-derived factor V/Va that is responsible for the observed protease resistance, and the mechanism by which Prothrombinase bound to platelets differs from a model system using vesicles composed of 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS), PCPS vesicles. Previous studies have demonstrated that activated platelets release a dissociable pool of factor V/Va and a non-dissociable, membrane-bound pool, which is covalently attached to the platelet membrane through a glycosylphosphatidyl inositol (GPI) anchor. Data described herein demonstrate unequivocally that the pool of platelet-derived factor V/Va that is resistant to proteolytic inactivation by activated protein C is provided exclusively by the non-dissociable GPI-anchored pool. Further, although this factor Va pool is susceptible to proteolysis by plasmin, the fragments formed are associated with sustained and increased cofactor activity. These observations indicate that tethering of factor Va to the membrane surface via a GPI anchor imparts resistance to proteolytic inactivation and sustained thrombin generation at sites of vascular injury. For several years it has been known that Prothrombinase assembled on PCPS vesicles does not mimic that bound to platelets. While both enzymes cleave prothrombin at Arg271 and Arg320 to form thrombin, prothrombin activation proceeds via the prethrombin-2 pathway (initial cleavage at Arg271) on the platelet surface, in contrast to the meizothrombin pathway (initial cleavage at Arg320) on PCPS vesicles. Using thrombin active site inhibitors, we demonstrate that the preference for either pathway is dictated by the conformation in which prothrombin is bound by the membrane-bound enzyme. The prethrombin-2 pathway of prothrombin activation catalyzed by platelet-bound Prothrombinase is a direct consequence of configuring prothrombin in a proteinase-like state resulting in the exposure of a pseudo-active site that can be stabilized by active site thrombin inhibitors. Conversely, prothrombin is preferentially configured in the zymogen-like state on PCPS vesicles where the meizothrombin pathway is preferred. Additional support for the differential assembly of Prothrombinase on the platelet surface is provided by observations made using prethrombin-1, an intermediate formed by cleavage of prothrombin at Arg155 by the formed thrombin. Prethrombin-1 is converted into fragment-2 and thrombin by platelet-bound Prothrombinase at a substantially higher rate than vesicle-bound Prothrombinase. The decreased rate of prethrombin-1 activation in the model system is due, in part, to inhibition of the vesicle-bound enzyme by the fragment-2 generated in the reaction. Taken together, these data not only provide important molecular insights into the mechanisms by which Prothrombinase bound to activated platelets at sites of vascular injury regulates the procoagulant response to effectively support robust thrombin generation, but also provides potential mechanistic sites that could be targeted therapeutically.