ALBERT

Description: The role of the intrinsic coagulation pathway in acute myocardial infarction is poorly defined. Both coagulation factors XII (FXII) and XI (FXI) support experimental thrombus propagation in animals. Additionally, humans with FXI deficiency have a lower incidence of thrombosis and stroke, however no such association has been established for FXII. Curiously, the incidence of previously verified myocardial infarction (MI) among 96 surviving FXI deficient subjects that were interviewed in an epidemiologic study was found to be similar to or possibly even higher than the recorded incidence of MI in an age/sex matched dataset from morbidity/mortality statistics of the general Israeli population (Salomon et al. J Thromb Haemost. 2003;1:658). However, the outcome of these coronary events were not reported, except for the fact that all interviewed FXI subjects were alive at the time of the interview. To investigate the contribution of FXI activation by FXIIa in experimental MI, we used a standard mouse model of acute myocardial ischemia (AMI). To inhibit FXI in the mouse, we utilized our monoclonal antibody (14E11) that targets the Apple 2 domain of FXI, and has been shown in vitro to inhibit the activation of FXI by factor XIIa, while not significantly inhibiting activation of FXI by thrombin. To evaluate the efficacy of 14E11 in reducing ischemic injury in mice, the left coronary artery (LCA) of wildtype male mice was reversibly ligated for 40 min, and 14E11 (1 mg/kg; iv) or vehicle was infused during the last 15 min of occlusion. Occlusion was confirmed by sustained S-T elevation, regional cyanosis and wall motion abnormalities. Following occlusion, the ligature was removed and the heart reperfused for 2 hr. To delineate the area of risk and ischemia, the LCA was re-occluded at 2 hr post-reperfusion and fluorescent polymers infused into the apex of the heart. The heart was excised, cut into 1 mm thick transverse slices and photographed under UV light to identify the area at risk. Tissue sections were additionally stained with 2,3,5-triphenyltetrazolium chloride solution and infarcted areas evaluated via morphometric analysis. The area at risk was evaluated as the percent of total heart volume and infarct size was calculated as the percentage of area at risk. Our results indicated that the area of risk did not differ between treatment groups, however treatment with 14E11 reduced infarct volume by 33% (p

Description: Abstract 5261 An increase in mean platelet volume (MPV) is correlated with platelet activation and subsequent shape change. Pathologic processes marked by increased platelet activity such as myocardial infarction, cerebral vascular accidents, diabetes mellitus, and hypertension are associated with an increased MPV. Elevated MPV in these conditions reflect both a higher level of platelet activation as well as increased platelet turnover secondary to platelet consumption within thrombus formation. Assessment of MPV can be used to risk stratify patients as well as assign them to prognostic categories. However, MPV does not assess platelet heterogeneity or the specific change in single platelet mass, volume, or density. Current methods provide little insight into changes in physical parameters at the single platelet level. In order to overcome this limitation, we developed a quantitative tomographic differential interference contrast (QTDIC) microscopy technique to measure dry mass, volume, and density of platelets at the single-cell level. This technique is based on determining the axially resolved refractive index from a series of through-focus DIC images. Single cell platelet mass was observed to reduce from 1.84 ± 0.14 pg to 1.60 ± 0.13 pg in response to stimulation with thrombin-receptor agonist peptide (TRAP), while single cell platelet volume reduced from 7.28 ± 0.56 fL to 6.03 ± 0.48 fL (mean ± SEM). Single cell platelet density increased from 0.25 ± 0.001 pg/fL to 0.26 ± 0.002 pg/fL (mean ± SEM). Taken together, we have characterized the physical parameters of platelets in response to agonist stimulation. Our data suggest that platelet activation may correlate with decreased mass and volume, perhaps as a consequence of platelet degranulation. Further elucidation of the morphological changes of activated platelets at the single platelet level may allow for better understanding of platelet function and dysfunction in patients affected by platelet granule deficiencies, giant platelet syndromes, and disorders associated with membrane receptorsFigure 1.Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density.Figure 1. Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1106 The plasma protease factor XIIa (FXIIa) contributes to vascular occlusion in murine thrombosis models, at least partly through activation of factor XI (FXI). While there is good correlation between plasma FXI levels and thrombotic events in humans, the situation is not as clear for FXII (the precursor of FXIIa), suggesting fundamental differences in thrombus formation in mice and humans. To facilitate studies on the effects of FXII/XIIa on thrombus formation, we developed novel inhibitory antibodies to human FXII, designated 9A2 and 15H8, by immunizing FXII-deficient mice with human FXII. Using recombinant human FXII molecules that lack various domains, and chimeras in which specific domains in FXII are replaced with those from the related protein hepatocyte growth factor activator, we determined that 9A2 and 15H8 bind to the FXII/XIIa non-catalytic heavy chain at different sites. 9A2 binds on or near the EGF2 domain, while 15H8 binds to the fibronectin type I and/or kringle domain. These areas have been implicated in FXII binding to polyanionic surfaces. Saturating concentrations of 9A2 or 15H8 reduced FXII activity by 50% and 90%, respectively, in an aPTT assay using normal plasma, while combining the antibodies resulted in 〉95% inhibition. However, in assays in which clot formation was triggered by adding FXIIa directly to plasma, preincubation of FXIIa with either antibody did not prolong the clotting time. Furthermore, neither antibody had a strong effect in a chromogenic assay of FXI activation by FXIIa, indicating the antibodies interfere with the aPTT assay primarily by inhibiting FXII activation. FXII activation in the aPTT assay is initiated by addition of a polyanion such as silica to the plasma to induce contact activation. In vivo, polymers of inorganic phosphate (polyP) may serve a similar function. Contact activation is triggered in plasma when FXII bound to the polyanion is activated, probably by trace amounts of FXIIa or another protease present in the plasma. Once formed, FXIIa converts the zymogens prekallikrein and FXI to the proteases kallikrein and FXIa, both of which can activate additional FXIIa to amplify the process. In the presence of 9A2 or 15H8, activation of pure FXII in the presence of either silica or polyP was significantly reduced. Interestingly, the antibodies actually potentiated FXII activation by kallikrein or FXIa in the absence of a polyanion. Taken as a whole, these results suggest that binding of 9A2 or 15H8 to FXII results in conformational changes that make FXII a better substrate for kallikrein and FXIa, possibly by mimicking the effect of FXII binding to a polyanion, but that prevent activation of FXII by FXIIa (autoactivation), blunting the overall rate of activation. We tested the effects of 9A2 and 15H8 in a mouse model in which thrombotic occlusion of the carotid artery is induced by exposing the vessel to a 3.5% solution of ferric chloride. Wild type C57Bl/6 mice develop arterial occlusion within 5 to 10 minutes, while FXII-deficient mice are resistant to arterial occlusion. Infusion of human FXII into FXII-deficient mice restores the wild type phenotype. 15H8 prevented thrombus formation in mice reconstituted with human FXII, while 9A2 reduced the rate of thrombotic occlusion by 50%. In an ex vivo flow model, perfusion of human blood through collagen-coated tubes at a shear rate of 300 sec−1 results in tube occlusion by platelet and fibrin rich clot in ∼15 minutes. 15H8 effectively blocked fibrin formation and reduced platelet accumulation, preventing tube occlusion. 9A2 was also effective at preventing clot formation, but there was evidence of some fibrin accumulation over time. In summary, the monoclonal anti-human FXII IgGs 9A2 and 15H8 prevent thrombus formation in whole blood in vivo and ex vivo by interfering with FXII activation. Our data support the hypothesis that pharmacologic inhibition of FXII activation may have therapeutic utility in disorders that are driven or aggravated by the blood contact system. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1060 Platelets serve as the primary mediators of hemostasis and thrombosis, circulating as surveyors for gaps in vascular integrity. As platelets encounter exposed extracellular matrix proteins, receptors on the platelet surface trigger intracellular signaling events that result in rapid platelet activation and a complex rearrangement of platelet morphology to form filopodia and lamellipodia. Rac1, a member of the Rho GTPase family, has emerged as a key regulator in platelet actin dynamics. However, the specific downstream events following Rac1 activation that mediate platelet actin cytoskeleton reorganization remain ill-defined. The Rho GTPase, Rac, supports the autocatalytic activation of the p21 activated kinases, or PAKs, to mediate actin reorganization processes in focal adhesion formation and cell migration. Upon activation by GTP-bound Rac, the PAKs phosphorylate a number of substrates to coordinate actin dynamics. Platelets express a number of PAK isoforms, and like Rac, PAK has been shown to be activated as platelets spread on collagen in a Src and PI3K dependent manner. Furthermore, the adaptor protein SLP-76 has been proposed to potentiate PAK activity downstream of Rac activation to mediate platelet lamellipodia formation. However, the specific roles of PAK in platelet function have yet to be characterized. Thus we set out to elucidate the role of PAK in platelet function and to define the connection between Rac activation, PAK, and platelet cytoskeletal reorganization. Our initial experiments with mass spectrometry revealed that following platelet activation, Rac1 associates with a set of PAK effectors, GIT1, GEFH1, LIMK1, and Merlin. We next demonstrated a co-localization of Rac1 and PAK with actin at the leading edge of spread platelets on fibrinogen. In addition, inhibition of PAK signaling by two different pharmacologic inhibitors blocked platelet focal adhesion and lamellopodia formation on both fibrinogen and collagen. Inhibition of PAK signaling abrogated intracellular calcium mobilization in platelets, prevented platelet aggregation to the GPVI-agonist, CRP, and destabilized platelet lamellipodia, resulting in the retraction of lamellipodia in spread platelets. Finally, inhibition of PAK resulted in the disaggregation of platelet aggregates formed under shear flow conditions. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation to PAK effector function and platelet lamellopodia formation, thus filling an important gap in the understanding of platelet actin cytoskeletal organization. In addition, these data characterize the integral role of PAK in platelet spreading, aggregation, and aggregate stability. Elucidating the mechanisms that mediate platelet spreading and aggregate formation may highlight important steps in the platelet activation cascade at which to pharmacologically intervene in order to inhibit or treat pathologic thrombi formation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3362 Activation of coagulation factors (F) XII and XI support thrombogenesis through multiple pathways. FXII-deficient mice are more resistant to FeCl3-induced arterial occlusion than either FIX or FXI deficient mice, suggesting that the resistance of FXII-deficient mice to experimental thrombosis is not completely explained by the FXII-FXI-FIX pathway, suggesting the existence of a pathological FXII by-pass, in vivo. The APTT of FXII deficient plasma is longer than the APTT of FXI, FIX, or FX deficient plasmas. We found that addition of 150 nM activated FXII (FXIIa) decreased the recalcification time of FXI or FIX-deficient plasma by up to 85%. In a purified system FXIIa could activate prothrombin but not FX. Addition of rivaroxaban, a FXa inhibitor, to FXI or FIX-deficient plasma blocked the observed procoagulant effect of FXIIa, suggesting that FXIIa promotes the activation of FX independent of FXI or FIX, but the ability of FXIIa alone to induce coagulation is insufficient in plasma, in vitro. Addition of long polyphosphate (polyP), typically found in bacteria, but not short polyP, which is secreted by activated platelets, decreased the recalcification time of FXI or FIX-deficient plasma. The presence of either corn trypsin inhibitor (CTI), that inhibits FXIIa, or rivaroxaban blocked the procoagulant effect of long polyP, suggesting that the activation of FXII by long polyP promotes coagulation in an FXI- and FIX-independent manner. Addition of CTI or an antibody that inhibits FIX activation by FXIa, but not addition of an antibody that inhibits activation of FXI by FXIIa, increased the time of occlusive thrombus formation in recalcified human blood that was driven through collagen and tissue factor (TF)-coated capillary tubes, consistent with the thrombogenic roles of FXIIa and feedback activation of FXI. Only CTI inhibited the prothrombotic effect of long polyP, also suggesting that FXIIa could be thrombogenic independent of FXI and FIX. In summary, we propose that pathological FXII activation, e.g., by foreign surfaces or long polyP, is thrombogenic both in FXI/FIX-dependent and -independent manners. Provided that FXII has no significant physiological function in humans, our data support the hypothesis that inhibition of FXII activity or activation may have safe antithrombotic effects. Disclosures: Morrissey: No organization, but the speaker is co-inventor on pending patent applications on the medical uses of polyphosphate: Patents & Royalties.

Description: Abstract 634 Metastatic cancer is associated with a hypercoagulable state, and pathological venous thromboembolic disease is a significant source of morbidity and the second leading cause of death in patients with cancer. Patients with cancer have a 4–10 fold increased risk of developing thrombosis. Recurrent thrombosis can be clinically managed with anticoagulant therapy; however, the risk of bleeding complications associated with the use of anticoagulants has prevented routine prophylactic anticoagulation for patients with cancer who have not yet developed thrombosis. Therefore, a method to identify which cancer patients are at imminent risk to develop thrombosis would allow for an objective means by which to administer personalized anticoagulant prophylaxis, reducing the morbidity and mortality for patients with cancer. There is currently a lack of laboratory assays capable of identifying which patients with cancer are at risk of developing thrombosis. Here we aimed to develop a novel labeling strategy to detect and quantify procoagulant circulating tumor cells (CTCs) from patients with metastatic cancer. We hypothesize that the enumeration of procoagulant CTCs may be prognostic for the development of venous thrombosis in patients with cancer. In this study, we characterized the binding of fluorescently-labeled active site-inhibited factors VIIa, Xa and IIa to the metastatic breast cancer cell line, MDA-MB-231, the non metastatic colorectal cell line, SW480, or the metastatic colorectal cell line, SW620, either in a purified system, in plasma, or in whole blood. Using flow cytometry, labeling of cancer cells in a purified system showed cell and factor-specific characteristics for labeling efficacy. Our data show that a concentration of 50 nM FVIIa-based probe was sufficient to label both the MDA-MB-231 and SW620 cells, while a concentration of 500 nM of the FXa- or FIIa-based probes was required to label both MDA-MB-231 and SW620 cells. A concentration of 0.5 μM FVIIa, 5 μM FXa and 5 μM FIIa was shown to label MDA-MB-231 and SW620 cells in anticoagulated plasma and in plasma under conditions of coagulation. We designed a series of experiments to determine whether our labeling strategy was amenable to a cell processing protocol that utilizes cancer cells being plated onto glass slides. We immobilized MDA-MB-231, SW480, and SW620 cells on functionalized glass surfaces and exposed them to fluorescently labeled FVIIa (0.5 μM), FXa (5 μM), FIIa (10 μM). We found that in a purified system, the MDA-MB-231 cells were robustly labeled with the FVIIa and FXa probes. The FVIIa and FXa probes weakly labeled the SW480 cells and SW620 cells. The FIIa probe failed to label any of the three cell lines. Under conditions of coagulation, the FVIIa probe labeled all the adherent MDA-MB-231 cells. Heterogeneous FVIIa-labeling was observed for both the SW480 and SW620 cell lines, with some of the adherent cells labeling brightly, while other cells on the same slide were not labeled by the FVIIa-probe. The FXa probe showed complete labeling of all three cell lines. The FIIa-probe showed complete labeling of the MDA-MB-231 and SW620 cell lines and heterogeneous labeling of the SW480 cells under conditions of coagulation. In whole blood, the FVIIa probe showed heterogeneous labeling of the MDA-MB-231 cells, SW480 and SW620 cells. Heterogeneous labeling of all three cell lines with the FXa and FIIa probes was observed, with very few SW480 or SW620 cells labeled. All three cell lines were labeled with an anti-TF mAb. In summary, we demonstrated the use of fluorescently-modified, active site-inhibited coagulation factors to label procoagulant cancer cells. We demonstrated that coagulation factors based-probes bound to cancer cell lines in purified systems and in whole blood, yet failed to bind to peripheral blood cells. Labeling of cancer cells was demonstrated via flow cytometry in purified systems, as well as on an immobilized-cell platform similar to what is currently used in some CTC-detection platforms. This work is the first step in the development of a function-based CTC labeling strategy to determine whether CTCs are procoagulant, and whether CTC enumeration and procoagulant characterization strategies are clinically useful in predicting thrombosis in patients with cancer. Disclosures: No relevant conflicts of interest to declare.

Description: The vasculature is a dynamic environment in which blood platelets constantly survey the endothelium for sites of vessel damage. The formation of a mechanically coherent hemostatic plug to prevent blood loss relies on a coordinated series of ligand–receptor interactions governing the recruitment, activation, and aggregation of platelets. The physical biology of each step is distinct in that the recruitment of platelets depends on the mechanosensing of the platelet receptor glycoprotein Ib for the adhesive protein von Willebrand factor, whereas platelet activation and aggregation are responsive to the mechanical forces sensed at adhesive junctions between platelets and at the platelet–matrix interface. Herein we take a biophysical perspective to discuss the current understanding of platelet mechanotransduction as well as the measurement techniques used to quantify the physical biology of platelets in the context of thrombus formation under flow.

Description: Introduction: Nearly 40% of adolescent women experience heavy menstrual bleeding (HMB), and identifiable bleeding disorders are diagnosed in only 20-60% of these patients. We suspect that qualitative platelet disorders contribute to HMB, but are under-diagnosed. A pilot study was conducted to evaluate platelet function in adolescent women with HMB employing four novel, small-volume, whole blood platelet function assays. In addition, primary and secondary hemostasis, bleeding phenotype, and quality of life were assessed. Methods: Patients referred to the Young Women's Hematology Clinic at Oregon Health & Science University for evaluation of HMB were offered participation in the study. Participants underwent standard review of their medical and family history and physical exam. Standard lab evaluation included CBC, PT, PTT, fibrinogen, thrombin time, Von Willebrand Panel, PFA-100, and iron studies with platelet aggregation or phenotyping performed if clinically indicated. Using less than 0.5 mL of whole blood, platelet function was assessed with four novel platelet function assays: assessment of platelet activation, secretion, and aggregation was assessed by flow cytometry analysis, while platelet adhesion and aggregation was assessed under shear in a capillary tube. Quality of life (QOL) was assessed using the PedsQL tool. Bleeding phenotype was assessed with the ISTH Bleeding Assessment Tool (ISTH BAT). Menorrhagia was assessed with the Pictorial Bleeding Assessment Chart (PBAC), the Philipp Tool and the clinical history. Results: Nine participants have enrolled on study to date, with 2 completing the 3-month visit. The median age of the cohort was 16 years (14-18 years). Eight out of nine categorized their period as heavy, 6 also had epistaxis, and 7 reported excessive bruising. The median ISTH BAT score was 4 (3-7). Of the 7 patients who had a Philipp Score obtained, 5 were positive. Median PBAC score was 161 (64-196). Median ferritin was 13 ng/mL (4-65 ng/mL). Median QOL psychosocial score was 70 (68.36-88.25), comparable to that of pediatric patients with cancer. Of the 9 participants, 6 had platelet aggregation and phenotyping. Four participants did not receive a bleeding disorder diagnosis, 1 was diagnosed with Type 1 VWD, 1 was diagnosed with bleeding disorder, NOS, and 1 was diagnosed with Ehlers Danlos Syndrome. Two participants were diagnosed with a qualitative platelet disorder (QPD): one based on platelet aggregation and one based on thromboelastography. The four novel platelet function assays confirmed platelet function abnormalities in the participants diagnosed with QPD's (Figure 1&2). Impaired platelet response to agonist stimulation was also observed in participants with non-platelet disorder bleeding disorder diagnoses and in participants without a bleeding disorder diagnosis. Conclusions: In this pilot study, the etiology of HMB in adolescent women was evaluated with four novel platelet assays in addition to standard assays of hemostasis. A bleeding disorder diagnosis was not made with standard evaluations in 4 out of 9 participants. The novel assays detected platelet abnormalities not observed using currently available clinical labs, and confirmed the presence of abnormal platelet function in participants with abnormal platelet function testing. These assays require significantly less blood volume than currently available assays and expand investigation of platelet function to platelet adhesion and platelet interactions in whole and flowing blood. Further work is needed to determine the sensitivity and specificity of the novel assays in detecting platelet dysfunction. Continued investigation into the impact of HMB on the adolescent female population is needed. Disclosures Haley: CSL Behring: Honoraria; Baxalta: Membership on an entity's Board of Directors or advisory committees. Recht:Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Genentech: Research Funding; Novo Nordisk: Research Funding; Baxalta: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Description: Background: ADAMTS13, a plasma metalloprotease, is secreted into blood as an active enzyme that cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular flow. Plasma ADAMTS13 has a molecular weight of 200 kDa, consisting of a metalloprotease (MET) domain, a disintegrin-like domain, a first thrombospondin type-1 repeat (TSP1) domain, a Cys-rich domain, and a spacer domain. Moreover, the C-terminal domain of ADAMTS13 contains an additional seven TSP1 repeats and two CUB domains. ADAMTS13 has been shown to adopt a natural folded conformation, allowing its CUB domains to interact with its spacer domain. This more closed conformation prohibits the functional exosite on the spacer domain from interacting with its proteolytic site on the A2 domain of VWF. In plasma, globular ADAMTS13 will associate with VWF via necessary binding of the CUB domains to the VWF D4CK fragment. Under shear stress or flow conditions, bound ADAMTS13 will unfold leading to exposure of the spacer domain exosite and ultimately increased ADAMTS13 proteolysis VWF. Without the CUB domains, ADAMTS13 does not proteolyze VWF under flow conditions. To date, it is still uncertain how ADAMTS13 activity is regulated, and what impact this has on the inactivation of VWF. The serine proteases thrombin, activated FX (FXa), and plasmin have been shown to cleave and inactivate ADAMTS13. Based on the fact that congenital factor XI deficiencies are associated with bleeding disorders and that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that the serine protease activated FXI (FXIa) inactivates ADAMTS13 leading to platelet aggregation and thrombus formation. Aim: To determine whether FXIa is able to cleave and inactivate ADAMTS13. Methods and results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for selected times (0-3 hours) at 37oC before being separated by SDS-PAGE and analyzed by Coomassie blue staining, resulting in the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of lower molecular weight bands under reducing conditions. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13 by FXIa. Samples were analyzed by western blot to determine the cleavage site using an anti-ADAMTS13 antibody, which specifically binds the two CUB domains, and an anti-ADAMTS13 antibody which specifically binds the MET domain. ADAMTS13 has been shown to be cleaved by serine proteases, such as plasmin, thrombin and FXa. We incubated ADAMTS13 (200nM) with equivalent concentrations of plasmin, thrombin, FXa, FXIa, FXIIa or Kallikrein at 37oC over a time interval of 0-3 hours. The addition of Ca++(5 mM) was necessary for proteolytic activity of thrombin and FXa. We observed that the ability of FXIa to cleave ADAMTS13 was found to be similar to the ability of thrombin to cleave ADAMTS13. Neither FXa, kallikrein, nor FXIIa appeared to cleave ADAMTS13. The antibody against the MET domain detected a single broad band at approximately 150 kDa. When the samples were analyzed with the antibody specific for the two CUB domains, a single broad band at approximately 50 kDa was detected, suggesting that the proteolysis of ADAMTS13 by FXIa preferentially occurs near the start of the first CUB domain. Interestingly, it has been previously reported that the CUB domains are necessary for VWF strand cleavage under flow conditions. It has been shown that the cleavage of a fluorescence-quenching substrate, FRETS-VWF73, by ADAMTS13 was enhanced after CUB1-2 domain removal. We observed that after the incubation of ADAMTS13 (30nM) with FXIa (30nM) at 37oC for 3 hours, the activity of ADAMTS13 was increased. Analysis of the samples by western blot using an anti-ADAMTS13 MET antibody confirmed the generation of the 150 kDa fragment. Conclusion: Our study suggests a novel molecular link between the regulation of VWF activity and FXI through inactivation of ADAMTS13. The results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also through limiting ADAMTS13-mediated VWF inactivation. Our future studies are focused on determining the physiological role of the proteolytic removal of the CUB domains of ADAMTS13 by FXIa under flow conditions by measuring platelet aggregation. Disclosures No relevant conflicts of interest to declare.