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A system for vessel characterization aimed at detecting deep vein thrombosis (DVT) in the lower limbs has been developed and evaluated using ultrasound image processing, location and force sensors measurements, blood flow information and a protocol based on the current clinical standard, compression ultrasound. The goal is to provide an objective and repeatable system to measure DVT in a rapid and standardized manner, as this has been suggested in the literature as an approach to improve overall detection of the disease. The system uses a spatial Kalman filter-based algorithm with an elliptical model in the measurement equation to detect vessel contours in transverse ultrasound images and estimate ellipse parameters, and temporal constant velocity Kalman filters for tracking vessel location in real-time. The vessel characterization also comprises building a 3-D vessel model and performing compression and blood flow assessments to calculate measures that indicate the possibility of DVT in a vessel. A user interface designed for assessing a vessel for DVT was also developed. The system and components were implemented and tested in simulations, laboratory settings, and clinical settings. Contour detection results are good, with mean and rms errors ranging from 1.47-3.64 and 3.69-9.67 pixels, respectively, in simulated and patient images, and parameter estimation errors of 5%. Experiments showed errors of 3-5 pixels for the tracking approaches. The measures for DVT were evaluated, independently and integrated in the system. The complete system was evaluated, with sensitivity of 67-100% and specificity of 50-89.5%. System learnability and memorability were evaluated in a separate user study, with good results. Contributions include a segmentation approach using a full parameter ellipse model in an extended Kalman filter, incorporating multiple measurements, an alternate sampling method for faster parameter convergence and application-specific initialization, and a tracking approach that includes a sub-sampled sum of absolutes similarity calculation and a method to detect vessel bifurcations using flow data. Further contributions include an integrated system for DVT detection that can combine ultrasound B-mode, colour flow and elastography images for vessel characterization, a system interface design focusing on usability that was evaluated with medical professionals, and system evaluations through multiple patient studies.

SAMMANFATTNING Bakgrund: PICC är en central infart som används inom vården för att kunna ge kärlretande läkemedel. Komplikationer vid användning av PICC kan vara infektion, trombos, tromboflebit eller stopp i katetern.  Syfte: Syftet med studien är att undersöka förekomsten av komplikationer av PICC hos onkologiska patienter samt för att se om det finns några skillnader mellan olika diagnosgrupper och behandlingar med avseende på förekomsten av djupa ventromboser (DVT) och infektioner. Syftet är också att ta reda på hur länge en PICC sitter och hur vanligt det är att en PICC felplaceras.  Metod: Metoden som används är en retrospektiv, deskriptiv, kvantitativ undersökning. I studien ingår alla onkologpatienter som fått en PICC år 2009-2011 (n=677). Data samlades in med hjälp av journalgranskning.  Resultat: Förekomsten av DVT var 5,6 %. Patienter som fick behandling med Capecitabin hade statistiskt signifikant mer DVT jämfört med andra behandlingar. Patienter som fick behandling med R-CHOP hade statistiskt signifikant mindre DVT jämfört med andra behandlingar. Antalet infektioner var 3 %. Stopp i katetern drabbade 1,8 % av patienterna, 17 % hade besvär med rodnad under förbandet, 12 % av alla katetrar åkte ut 4 cm eller mer och 2,5 % av katetrarna felplacerades vid inläggningen. En PICC var insatt i medelvärde 92 dagar, median 105 dagar.  Slutsats: Förekomsten av komplikationer av PICC var låg hos onkologiska patienter med undantag för hudbesvär som förekom hos var sjätte patient. Behandlingar innehållande Capecitabin förefaller öka risken för DVT men fler studier behövs för att öka kunskaperna om detta. PICC är en säker venös infart vid behandling med cytostatika. / ABSTRACT Background: PICC (peripherally inserted central catheter) is a central line used in healthcare to provide vascular irritant drugs. Complications with PICC can be infection, thrombosis, thrombophlebitis or occlusion of the catheter.  Aim: The aim of the study is to examine the incidence of complications of PICC in oncology patients and to see if there are any differences between diagnostic groups and treatments for the presence of deep venous thrombosis (DVT) and infection. The aim is also to find out for how long time a PICC is inserted and how common it is for a PICC misplaced.  Method: The method used is a retrospective, descriptive, quantitative survey. The study includes all oncology patients who received a PICC years 2009-2011 (n=677). Data were collected through medical record review.  Results: The incidence of DVT was 5,6 %. Patients treated with Capecitabin had statistically significantly more DVT compared with other treatments. Patients treated with R-CHOP had statistically significantly less DVT compared with other treatments. The incidence of infections was 3 %. Occlusion of the catheter affected 1,8 % of patients, 17 % had problems with redness under the dressing, 12 % of all catheters went out four cm or more and 2,5 % of the catheters were misplaced at insertion. A PICC was inserted in mean 92 days, median 105 days.  Conclusion: The complication rate of PICC was low in oncology patients with the exception of skin problems that occurred in every sixth patient. Treatments containing Capecitabin appears to increase the risk of DVT but more studies are needed to raise awareness of this. PICC is a safe venous access for chemotherapy.

Von Willebrand factor (VWF) promotes platelet adhesion and aggregation at sites of vascular damage. This function is directly related to the multimer size of VWF. The VWF-specific metalloprotease ADAMTS13 decreases VWF multimer size by cleaving at Y1605-M1606 in the VWF A2 domain. This thesis examined the sensitivity of ADAMTS13 cleavage to mutagenesis of the full-length multimerized VWF substrate, and a small VWF A2 domain fragment, VWF115. The ADAMTS13 cleavage site at Y1605-M1606 was mutated with the most severe loss of cleavage observed in Y1605A/M1606A. In addition, 4 single nucleotide polymorphisms were examined, with D1472H, Q1571H, P1601T proteins all showing increased resistance to cleavage. In contrast, G1643S has enhanced cleavage in the full-length VWF substrate but shows cleavage resistance in VWF115. Three von Willebrand disease mutations were also examined. In patients, R1597W has enhanced ADAMTS13 cleavage and a loss of high molecular weight multimers, while R1205H has enhanced protein clearance resulting in very low VWF levels and Y1584C patients have moderately low VWF levels. R1597W has enhanced cleavage of full-length VWF, while a slight cleavage increase is observed in VWF115 for Y1584C, and no change is seen with R1205H. The VWF mutations R1597W, Y1605A/M1606A, R1205H and Y1584C were further examined in the VWF knockout mouse using recombinant VWF protein infusion and hydrodynamic delivery of VWF cDNA to determine the effects these mutations produce on VWF antigen levels, multimer structure, secretion, clearance and function in a thrombotic injury model. All four mutations had different pathogenic mechanisms. R1597W showed accelerated clearance with loss of multimer structure, and greatly increased time to thrombotic occlusion. Y1605A/M1606A showed accelerated clearance with normal or supranormal multimer structure, a loss of thrombotic occlusion but increased platelet accumulation. Y1584C showed no change in protein clearance, with decreased VWF antigen level, reduced multimer structure, and reduced thrombotic potential. R1205H demonstrated a synthetic defect in vitro and in vivo increased clearance with a decrease in VWF antigen levels and normal multimer structure and a variable thrombotic potential. These results validate the use of the genetically-modified VWF knockout mouse model for evaluating the pathogenic mechanisms of putative VWF mutations. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-07-28 10:24:40.654

Le CD40 ligand (CD40L) est une molécule inflammatoire appartenant à la famille du Facteur de Nécrose Tumorale ("Tumor Necrosis Factor", TNF), originalement identifié au niveau des cellules immunitaires. L’interaction du CD40L avec son récepteur de haute affinité présent sur les cellules B, le CD40, est d’une importance cruciale à la production d’immunoglobulines lors de la réponse immunitaire. Aujourd’hui, nous savons que ces deux molécules qui constituent l’axe CD40/CD40L sont aussi exprimées au niveau des cellules du système vasculaire et occupent une place importante dans une variété de réactions inflammatoires, de sorte que le CD40L est présentement reconnu comme une molécule thrombo-inflammatoire prédictive des évènements cardiovasculaires. Les plaquettes sont la principale source du CD40L soluble ("soluble CD40L", sCD40L) plasmatique et il fut démontré être impliqué dans l’activation plaquettaire, malgré que son impact exact sur la fonction plaquettaire et les mécanismes sous-jacents demeurent inconnus. Ainsi, le but de ce projet était de déterminer l’impact du sCD40L sur la fonction plaquettaire et d’élucider les mécanismes cellulaires et moléculaires sous-jacents. Les objectifs spécifiques étaient : 1) d’évaluer l’impact du sCD40L sur l’activation et l’agrégation plaquettaire in vitro; 2) de déterminer le récepteur cible (CD40 ou autre) impliqué dans ces effets; 3) de décortiquer les voies signalétiques intracellulaires et moléculaires induites par le sCD40L, impliquant la participation potentielle de la famille du facteur associé du récepteur du TNF ("Tumor Necrosis Factor Receptor Associated Factor", TRAF) et 4) d’analyser l’effet du sCD40L sur la formation du thrombus in vivo. Le sCD40L augmente fortement l’activation et l’agrégation plaquettaire induite par de faibles doses d’agonistes. Les plaquettes humaines traitées avec une forme mutante du sCD40L qui n’interagit pas avec le CD40 et les plaquettes de souris CD40 déficientes (CD40-/-) ne furent pas en mesure d’induire ces effets. De plus, nous démontrons la présence de plusieurs membres de la famille des TRAFs dans les plaquettes, parmi lesquels seulement TRAF-2 interagit avec le CD40 suite à la stimulation par le sCD40L. Le sCD40L agit sur les plaquettes au repos par l’entremise de la protéine Rac1 et de sa cible en aval, soit la protéine kinase activatrice du mitogène p38 ("Mitogen Activating Protein Kinase", MAPK). Ceci mène ultimement au changement de forme plaquettaire et à la polymérisation de l’actine. Par ailleurs, il est intéressant de noter que les souris CD40-/- démontrent un défaut significatif de l’agrégation plaquettaire en réponse au collagène, ce qui souligne l’importance du CD40 dans les interactions plaquettes-plaquettes. Dans un deuxième temps, le sCD40L amplifie l’agrégation plaquettaire en sang complet, accélère les temps de thrombose in vitro mesurés à l’aide du système PFA-100 et augmente l’adhésion plaquettaire au collagène sous condition de flux, le tout par l’entremise du CD40. Finalement, dans un modèle de thrombose artérielle murin, l’infusion du sCD40L exacerbe la formation du thrombus chez les souris du type sauvage ("Wild Type", WT), mais non chez les souris CD40-/-. Ceci fut en plus associé à une augmentation significative du nombre de leucocytes au sein du thrombus des souris WT traitées à l’aide du sCD40L, tel que démontré par marquage immuno-histologique anti-CD45 et par quantification des coupes artérielles par microscopie optique. En résumé, ce projet identifie une nouvelle voie signalétique, TRAF-2/Rac1/p38 MAPK, en réponse au sCD40L et démontre ses effets sur l’activation et l’agrégation plaquettaire. De manière encore plus importante, nous démontrons pour la première fois la présence d’une corrélation positive entre les niveaux circulants du sCD40L et la thrombose artérielle, tout en soulignant l’importance du CD40 dans ce processus. Ainsi, le sCD40L constitue un activateur important des plaquettes, les prédisposant à une thrombose exacerbée en réponse au dommage vasculaire. Ces résultats peuvent expliquer le lien étroit qui existe entre les niveaux circulants du sCD40L et l’incidence des maladies cardiovasculaires. / CD40 ligand (CD40L) is an inflammatory molecule of the tumor necrosis factor (TNF) superfamily originally identified on cells of the immune system. Interaction of CD40L with its respective receptor on B cells, CD40, is of critical importance for immunoglobulin isotype switching during the immune response. Today, we know that these two molecules are also present on cells of the vascular system and have important implications in various inflammatory reactions, such that CD40L is now regarded as a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L) found in plasma and it has been shown in return to influence platelet activation, albeit the exact functional impact and underlying mechanisms remain undefined. Hence, this project was designed to investigate the effects of sCD40L on platelet function and to elucidate the cellular and molecular mechanisms involved. The specific aims of this study are four fold: 1) evaluate the impact of sCD40L on platelet activation and aggregation in vitro; 2) determine through which receptor (CD40 or other) these responses are mediated; 3) elucidate the underlying intracellular signalling pathways and molecular mechanisms involved, including the potential participation of the Tumor Necrosis Factor Receptor Associated Factor (TRAF) family and 4) assess the impact of sCD40L on thrombus formation in vivo. sCD40L strongly enhances activation and aggregation of washed human platelets induced by sub-threshold concentrations of agonists. Human platelets treated with a mutated form of sCD40L that does not bind CD40 and CD40 deficient (CD40-/-) mouse platelets failed to elicit such responses. Furthermore, we found that platelets express multiple members of the TRAF family, among which only TRAF-2 associates with CD40 upon sCD40L stimulation. Noticeably, sCD40L primes resting platelets through activation of the small GTPase Rac1 and its downstream target p38 mitogen activating protein kinase (MAPK), which leads to platelet shape change and actin polymerization. Interestingly, CD40-/- mice exhibit impaired platelet aggregation in response to collagen, thus highlighting the importance of CD40 in platelet-platelet interactions. Moreover, sCD40L dose-dependently enhances whole blood platelet aggregation and accelerates platelet function analyzer (PFA)-100 closure times in a CD40-dependant fashion. Preincubation of whole blood with sCD40L significantly increases platelet adhesion to collagen in an ex-vivo perfusion system under flow. In addition, in a ferric chloride-induced murine arterial thrombosis model, infusion of sCD40L exacerbates thrombus formation in wild type (WT), but not in CD40-/- mice. This was further associated with increased leukocyte infiltration within the thrombus mass of WT mice treated with sCD40L. In this project, we identify a new TRAF-2/Rac1/p38 MAPK signaling pathway in response to sCD40L and its effects on platelet activation and aggregation. More importantly, we establish a direct positive correlation between levels of sCD40L and thrombus formation in vivo, while highlighting the requirement of CD40 in this process. Thus, sCD40L is an important platelet primer predisposing platelets to enhanced thrombus formation in response to vascular injury. These results may explain the link between circulating levels of sCD40L and the occurrence of cardiovascular diseases.

I assessed the accuracy of two clinical prediction rules, the d-dimer blood test and point of care ultrasound for diagnosing lower limb deep vein thrombosis. Emergency physicians were trained in ultrasound and prospectively scanned emergency department patients with suspected deep vein thrombosis. Accuracy of the Wells and AMUSE rules and the ultrasound result was compared to radiology-performed ultrasound and a 90-day clinical outcome. Univariate and multivariate analyses were performed assessing which factors were associated with the outcome. The sensitivity and specificity of the Wells score for the clinical outcome was 85.7% and 68.5%; the AMUSE score 85.7% and 54.4%. Ultrasound had a sensitivity of 91.7% and specificity of 91.7% for radiology-diagnosed thrombus and 78.6% and 95.0% for clinical outcome. The odds ratio of a positive outcome with a positive ultrasound was 65.1. After receiving the ultrasound training program, emergency physicians were unable to demonstrate sufficient accuracy to replace current diagnostic strategies.

Anti-Beta2-Glycoprotein I antibodies (anti-b2GPI) are strongly associated with thrombosis in patients with primary antiphospholipid syndrome (PAPS). Anti-b2GPI activate endothelial cells (EC) resulting in a pro-thrombotic and pro-inflammatory phenotype. In order to characterise EC gene regulation in response to anti-b2GPI, early global gene expression was assessed in human umbilical vein endothelial cells (HUVEC) in response to affinity purified anti-b2GPI. Sera were collected from patients with PAPS and IgG was purified using HiTrap Protein G Sepharose columns. Polyclonal anti-b2GPI were prepared by passing patient IgG through NHS activated sepharose coupled to human b2GPI. Anti-b2GPI preparations were characterized by confirming their b2GPI co-factor dependence, binding to b2GPI and ability to induce leukocyte adhesion molecule expression and IL-8 production in vitro. Two microarray experiments tested differential global gene expression in 6 individual HUVEC donors in response to 5 different PAPS polyclonal anti-b2GPI (50 mg/ml) compared to 5 normal control IgG (50 mg/ml) after 4 hours incubation . Total HUVEC RNA was extracted and cRNA was prepared and hybridised to Affymetrix HG-133A (Exp.1) and HG-133A_2 (Exp.2) gene chips. Data were analyzed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. Significant change in gene expression was defined as greater than two fold increase or decrease in expression (p<0.05). Novel genes not previously associated with PAPS were induced including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1 and growth factors, CSF2, CSF3, IL-6, IL1b and FGF18. Downregulated genes were transcription factors/signaling molecules including ID2. Microarray results were confirmed for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C) using quantitative real-time RT-PCR analysis. This study revealed a complex anti-b2GPI-regulated gene expression profile in HUVEC in vitro. The novel chemokines and pro-inflammatory cytokines identified in this study may contribute to the vasculopathy associated with PAPS.

All artificial organ systems and medical devices that operate in direct contact with blood elicit activation of coagulation and platelets, and their long-term use often necessitates antithrombotic therapies that carry significant cost and bleeding risk. Thrombomodulin (TM) is a major endogenous inhibitor of blood coagulation localized on the endothelial cell surface. The overall objective of this research is to develop clinically durable synthetic materials by incorporating TM as a solid-supported film to actively and sustainably attenuate thrombus formation at the blood-contacting interface. During the course of this research, we developed site-specific approaches to covalently attach TM on the luminal surface of commercial vascular grafts using bioorthogonal chemistry that was compatible with ethylene oxide sterilization. Notably, we demonstrated the superior efficacy of TM to reduce platelet deposition compared with commercial heparin modified grafts using a non-human primate model of acute graft thrombosis. Finally, we optimized a novel reversible chemistry to rapidly and repeatedly regenerate immobilized TM, with the potential to significantly extend the lifetime of biologically active films.

Le CD40 est un membre de la famille des récepteurs du facteur de nécrose tumorale ("Tumour necrosis factor", TNF), initialement identifié sur des cellules de carcinome de la vessie. L'interaction du CD40 avec son ligand (CD40L) est d'une importance cruciale pour le développement des cellules B et de la commutation d'isotype au cours de la réponse immunitaire acquise. L'expression du complexe CD40/CD40L était initialement cru d'être limiter aux cellules du système immunitaire, mais aujourd'hui il est bien connu que ce complexe est également exprimé sur les cellules du système circulatoire et vasculaire, et est impliqué dans diverses réactions inflammatoires; de sorte que le CD40L est maintenant considéré comme une molécule thrombo-inflammatoire prédictive des événements cardiovasculaires. Les plaquettes expriment constitutivement le CD40, alors que le CD40L n'est exprimé que suite à leur l'activation. Il est ensuite clivé en sa forme soluble (sCD40L) qui représente la majorité du sCD40L en circulation. Il fut démontré que le sCD40L influence l'activation plaquettaire mais son effet exact sur la fonction plaquettaire, ainsi que les mécanismes cellulaires et moléculaires sous-jacents à son action demeurent inconnus. Ainsi, ce projet a été entrepris dans le but d’adresser les objectifs spécifiques suivants: 1) évaluer les effets in vitro du sCD40L sur l'activation et l'agrégation plaquettaire; 2) identifier les récepteurs plaquettaires impliqués dans l’action du sCD40L; 3) élucider les voies signalétiques intracellulaires induits par le sCD40L; 4) évaluer les effets du sCD40L sur la formation de thrombus in vivo. Nous avons trouvé que le sCD40L augmente fortement l'activation et l'agrégation des plaquettes en réponse à de faibles concentrations d'agonistes. Les plaquettes humaines traitées avec une forme mutante du sCD40L qui n'interagit pas avec le CD40, et les plaquettes de souris déficientes en CD40 ne furent pas en mesure d'induire de telles réponses, indiquant que le récepteur principal du sCD40L au niveau des plaquettes est le CD40. En plus, nous avons identifié la présence de plusieurs membres de la famille du facteur associé du récepteur du TNF ("TNF receptor-associated factor", TRAF) dans les plaquettes et nous avons montré que seulement le TRAF2 s'associe avec le CD40 suite à la stimulation par le sCD40L. Nos résultats indiquent aussi que le sCD40L agisse sur les plaquettes au repos par l'entremise de deux voies signalétiques distinctes. La première voie implique l'activation de la petite GTPase Rac1 et de sa cible en aval, soit la protéine kinase p38 activée par le mitogène ("p38 mitogen-activated protein kinase", p38 MAPK ), menant au changement de forme plaquettaire et à la polymérisation de l'actine; alors que la deuxième voie implique l'activation de la cascade signalétique du NF-kB. Par ailleurs, à la suite d'une lésion artérielle induite par le chlorure de fer, le sCD40L exacerbe la formation de thrombus et l'infiltration leucocytaire au sein du thrombus dans les souris du type sauvage, mais pas chez les souris déficientes en CD40. En conclusion, ce projet a permis d'identifier pour la première fois deux voies signalétiques distinctes en aval du CD40 plaquettaire et a permis d'établir leur implication dans l'activation et l'agrégation plaquettaire en réponse au sCD40L. De manière plus importante, ce projet nous a permis d'établir un lien direct entre les niveaux élevés du sCD40L circulant et la formation de thrombus in vivo, tout en soulignant l'importance du CD40 dans ce processus. Par conséquent, l'axe CD40/CD40L joue un rôle important dans l'activation des plaquettes, les prédisposant à une thrombose accrue en réponse à une lésion vasculaire. Ces résultats peuvent expliquer en partie la corrélation entre les taux circulants élevés du sCD40L et l'incidence des maladies cardiovasculaires. / CD40 is a member of the tumour necrosis factor (TNF) receptor family, originally identified on human bladder carcinoma cells. Interaction of CD40 with its ligand (CD40L) is of crucial importance for B cell development and immunoglobulin isotype switching during the adaptive immune response. Expression of the CD40/CD40L dyad was initially thought to be restricted to cells of the immune system, but today it is known to be also expressed on cells of the circulatory and vascular systems, and have important implications in various inflammatory reactions, such that CD40L is now regarded as a thrombo-inflammatory molecule and a reliable predictor of cardiovascular events. Platelets constitutively express CD40, whereas CD40L is expressed upon activation and subsequently cleaved into its soluble form (sCD40L), accounting for the majority of circulating sCD40L. Soluble CD40L has been shown to influence platelet activation but its precise effect on platelet function, and the underlying cellular and molecular mechanisms remain undefined; hence the purpose of this project. The specific aims of this study are: 1) to evaluate the in vitro effects of sCD40L on platelet activation and aggregation; 2) to determine the receptor(s) on platelets involved in the action of sCD40L; 3) to elucidate the intracellular signalling pathways induced by sCD40L; and 4) to evaluate the in vivo effects of sCD40L on thrombus formation. We have showed that sCD40L strongly enhances activation and aggregation of washed human platelets in response to sub-threshold concentrations of agonists. Human platelets treated with a mutated form of sCD40L that lacks CD40 binding, and platelets from CD40 deficient mice failed to elicit such responses, indicating that CD40 is the major platelet receptor for sCD40L. Moreover, we identified the presence of multiple members of the TNF receptor-associated factor (TRAF) in platelets and showed that only TRAF2 associates with CD40 after sCD40L stimulation. Interestingly, sCD40L primes resting platelets through two distinct signalling pathways. The first pathway involves activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, leading to platelet shape change and actin polymerization; whereas the second pathway involves activation of the NF-κB signalling cascade. Furthermore, sCD40L exacerbates thrombus formation and leukocyte infiltration within the thrombus mass in wild-type mice but not in CD40 deficient mice following ferric chloride-induced arterial injury. In conclusion, we have identified for the first time two distinct signalling pathways downstream of platelet CD40, and established their implication in platelet activation and aggregation in response to sCD40L. Noticeably, we established a direct link between elevated levels of sCD40L and in vivo thrombus formation, while emphasizing the requirement of CD40 in this process. Therefore, the CD40/CD40L dyad plays an important role in platelet priming that predisposes platelets to enhanced thrombus formation in response to vascular injury. These results may partly explain the correlation between elevated circulating levels of sCD40L and the incidence of cardiovascular diseases.

Atherosclerosis is a major cause of mortality in industrialized nations. Atherosclerosis is characterized by plaque deposition which decreases the lumen diameter into a stenosis. The creation of a restriction increases shear rates pathologic levels exceeding 3,500/s. Following plaque cap rupture, thrombus may form from the accumulation of millions of platelets, occluding the vessel, leading to heart attack and stroke. Studies of high shear thrombosis show that platelet activation, GPIIb/IIIa and vWF are involved. However, some recent studies also suggest that high shear aggregation is not dependent on activation or GPIIb/IIIa. Several antiplatelet pharmaceuticals against activation and GPIIb/IIIa have been proposed, but their efficacy in patients remains mixed. The overall objective of this project is to determine the factors necessary for thrombosis to occlusion in very high shear regions seen in diseased arteries. Our central hypotheses are that platelet activation and the subsequent conformational change in GPIIb/IIIa are necessary for thrombosis, and that higher concentrations of vWF in the plasma will increase thrombosis. To this end, we developed a new high shear hemodynamic model utilizing 30mLs of whole blood and quantified thrombus thickness, volume accumulation and accumulation rates. We demonstrate that thrombosis to occlusion stems from a second phase of Rapid Platelet Accumulation (RPA). Thrombus accumulation is completely prevented by PGE1 inhibition of platelet activation. Similarly, GPIIb/IIIa blockade via abciximab prevented significant thrombus deposition and RPA. We also found that increasing plasma vWF levels in high shear regions increased thrombus thickness and suggestively increased RPA rates. The results clarify the need for activation of mural platelets for long term thrombus accumulation without the activation of circulating platelets.