Platelet Deposition Research Articles

Simulation of thrombosis in a stenotic microchannel: The effects of vWF-enhanced shear activation of platelets

This study was undertaken to develop a numerical/computational simulation of von Willebrand Factor (vWF) - mediated platelet shear activation and deposition in an idealized stenosis. Blood is treated as a multi-constituent mixture comprised of a linear fluid component and a porous solid component (thrombus). Chemical and biological species involved in coagulation are modeled using a system of coupled convection-reaction-diffusion (CRD) equations. This study considers the cumulative effect of shear stress (history) on platelet activation. The vWF activity is modeled as an enhancement function for the shear stress accumulation and is related to the experimentally-observed unfolding rate of vWF. A series of simulations were performed in an idealized stenosis in which the predicted platelets deposition agreed well with previous experimental observations spatially and temporally, including the reduction of platelet deposition with decreasing expansion angle. Further simulation indicated a direct relationship between vWF-mediated platelet deposition and degree of stenosis. Based on the success with these benchmark simulations, it is hoped that the model presented here may provide additional insight into vWF-mediated thrombosis and prove useful for the development of more hemo-compatible blood-wetted devices in the future.

Links Between Complement Activation and Thrombosis

The coagulation and complement systems are central mediators of innate immune defense and aide the peri- and intravascular elimination of invading microorganisms in a process termed immuno-thrombosis. Complement and coagulation proteases are engaged in reciprocal amplifications at different levels of these enzymatic cascades. In addition, activated complement factor (C) 3 specifically stimulates platelets through C3a receptor signaling and thereby amplifies thrombus formation. A non-enzymatic function of complement activation emerged as a crucial mechanism that rapidly alters the function of the extrinsic coagulation activator tissue factor (TF) on monocytes. Activation of the classical complement pathway by therapeutic anti-thymocyte globulin preparations rapidly enhances monocyte TF procoagulant activity. On the one hand, complement activation-associated thiol-disulfide exchange supports protein disulfide isomerase-dependent conformational changes in TF. Insertion of the C5b-C7 complex supports these conformational changes in TF by exposing procoagulant phosphatidylserine (PS). Furthermore, these dual roles of complement activation play a central role in venous thrombosis. C3, but not C5-deficient mice are protected from platelet deposition. In contrast, C5 knock-out mice show diminished PS exposure on leukocytes and lack TF-dependent coagulation activation and fibrin deposition at the flow restricted venous vessel wall. Antiphospholipid antibodies (aPL) also cause TF- and complement-dependent thrombosis. Whereas C3-dependent thiol-disulfide exchange is again required for converting TF to a procoagulant form, aPL do not rely on downstream complement components for the exposure of PS and induce pathological thrombosis in C5-deficient mice. Our recent data show that aPL dissociate a cell surface-localized, inhibited complex with monocyte-expressed TF pathway inhibitor (TFPI). In addition to promoting TF procoagulant activation, complement-dependent thiol-disulfide exchange emerged as an additional central mechanism that allows for aPL internalization and endosomal signaling involving integrin- and TF cytoplasmic domain-dependent translocation of the NADPH oxidase to the endosome. A monoclonal antibody that traps TF with low procoagulant activity on the surface prevents TF endosomal trafficking, aPL signaling and aPL-induced pregnancy loss. These complement-dependent effects require assistance by thrombin-PAR1/PAR2 heterodimer signaling initiated by FXa dissociated from the inhibited TF complex. Monocyte TFPI-dysfunctional mice are protected from aPL pathogenic signaling in vitro and from aPL-induced thrombosis, but form thrombi normally in other experimental thrombosis models. These data show that formation of an inhibited TF cell surface complex specifically primes monocytes for thrombosis and pathogenic aPL signaling. Thus, the evolutionary conserved crosstalk of complement and coagulation cascades not only plays crucial roles in thrombosis, but also regulates thrombo-inflammatory signaling in autoimmune disease. Disclosures Ruf: MeruVasimmune: Equity Ownership; Iconic Therapeutics: Consultancy.

Surgical Techniques for Implanting the EVAHEART 2 Double Cuff Tipless Inflow Cannula.

The EVAHEART 2 (Sun Medical Technology Research Corporation, Nagano, Japan) is an investigational centrifugal ventricular assist device in the United States, introduced a new type inflow, named "double cuff tipless" inflow cannula intended to mitigate the risks of cannula malposition and subsequent ischemic stroke events associated with thrombi around the inflow cannula. To achieve these performance benefits of the "tipless" design, however, it is crucial to adhere to the recommended surgical procedure. We introduced a polymer-based patient model that mimics a dilated cardiomyopathy apex for inflow cannula implantation training. Here, we used the model to simulate appropriate and inappropriate techniques for inflow suturing. With the appropriate technique, the inflow ostium is aligned on the endocardial plane, and the cut myocardial surface is not exposed to the bloodstream. By contrast, with the inappropriate technique, which is represented as a worst-case suturing scenario, the inflow ostium is misaligned, thus exposing the cut myocardial cross-section to the bloodstream. This misalignment can predispose to platelet deposition, thrombus formation, and pannus formation with long-term support. Repeated training with this human apex model is important to confirm the inflow position and intraventricular finish before moving on to human cases. Along with rigorous preclinical training, technical adherence will help to ensure better clinical outcomes.

Von Willebrand factor delays liver repair after acetaminophen-induced acute liver injury in mice

Acetaminophen (APAP)-induced acute liver failure is associated with substantial alterations in the hemostatic system. In mice, platelets accumulate in the liver after APAP overdose and appear to promote liver injury. Interestingly, patients with acute liver injury have highly elevated levels of the platelet-adhesive protein von Willebrand factor (VWF), but a mechanistic connection between VWF and progression of liver injury has not been established. We tested the hypothesis that VWF contributes directly to experimental APAP-induced acute liver injury. Wild-type mice and VWF-deficient (Vwf-/-) mice were given a hepatotoxic dose of APAP (300 mg/kg, i.p.) or vehicle (saline). VWF plasma levels were measured by ELISA, and liver necrosis or hepatocyte proliferation was measured by immunohistochemistry. Platelet and VWF deposition were measured by immunofluorescence. In wild-type mice, VWF plasma levels, high molecular weight (HMW) VWF multimers, and VWF activity decreased 24 h after APAP challenge. These changes coupled to robust hepatic VWF and platelet deposition, although VWF deficiency had minimal effect on peak hepatic platelet accumulation or liver injury. VWF plasma levels were elevated 48 h after APAP challenge, but with relative reductions in HMW multimers and VWF activity. Whereas hepatic platelet aggregates persisted in livers of APAP-challenged wild-type mice, platelets were nearly absent in Vwf-/- mice 48 h after APAP challenge. The absence of platelet aggregates was linked to dramatically accelerated repair of the injured liver. Complementing observations in Vwf-/- mice, blocking VWF or the platelet integrin αIIbβ3 during development of injury significantly reduced hepatic platelet aggregation and accelerated liver repair in APAP-challenged wild-type mice. These studies are the first to suggest a mechanistic link between VWF, hepatic platelet accumulation, and liver repair. Targeting VWF might provide a novel therapeutic approach to improve repair of the APAP-injured liver. Patients with acute liver injury due to acetaminophen overdose have highly elevated levels of the platelet-adhesive protein von Willebrand factor. It is not known whether von Willebrand factor plays a direct role in the progression of acute liver injury. We discovered that von Willebrand factor delays repair of the acetaminophen-injured liver in mice and that targeting von Willebrand factor, even in mice with established liver injury, accelerates liver repair.

Overproduction of thrombopoietin by BRAFV600E‐mutated mouse hepatocytes and contribution of thrombopoietin to hepatocarcinogenesis

In hepatocarcinogenesis induced by diethylnitrosamine (DEN) in B6C3F1 mice, the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role. The livers of transgenic mice with a hepatocyte‐specific human BRAFV600E mutation weighed 4.5 times more than that of normal mice and consisted entirely of hepatocytes, resembling DEN‐induced preneoplastic hepatocytes. However, these transgenic mice spontaneously died 7 wk after birth, therefore this study aimed to clarify the causes of death. In the transgenic mice, the liver showed thrombopoietin (TPO) overexpression, which is associated with eventual megakaryocytosis and thrombocytosis, and activated platelets were deposited in hepatic sinusoids. TPO was also overexpressed in the DEN‐induced hepatic tumors, and sinusoidal platelet deposition was observed in the hepatic tumors of humans and mice. Podoplanin was expressed in some of the Kupffer cells in the liver of the transgenic mice, indicating that platelet activation occurred via the interaction of podoplanin with C‐type lectin receptor 2 (CLEC‐2) on the platelet membrane. Additionally, erythrocyte dyscrasia and glomerulonephropathy/interstitial pneumonia associated with platelet deposition were observed. In the transgenic mice, aspirin (Asp) administration prevented platelet activation, reduced the liver/body weight ratio, decreased the platelet deposition in the liver, kidney, and lung, and prevented erythrocyte dyscrasia and ameliorated the renal/pulmonary changes. Thrombopoietin overproduction by BRAFV600E‐mutated hepatocytes may contribute to hepatocyte proliferation via thrombocytosis, platelet activation, and the interaction of platelets with hepatic sinusoidal cells, while hematologic, renal, and pulmonary disorders due to aberrant platelet activation may lead to spontaneous death in the transgenic mice.

Abstract 5172: Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis

Abstract Platelets can activate hepatocyte proliferation not only via releasing the platelet factors, but also promoting for liver sinusoidal endothelial cells (LSEC) and Kupffer cells to produce the factors such as IL-6 and TGF-α that are indispensable for hepatocyte proliferation. We previously reported that in the transgenic (TG) mice with liver specific human BRAFV600E mutation, which corresponds to the mouse BrafV637E that is highly prevalent in mouse DEN-induced hepatic tumors, exhibit increased liver weight five times larger than that of normal mice. Furthermore, hepatocytes in these mice overexpress thrombopoietin (TPO) in association with megakaryocytosis/thrombocytosis, and large numbers of platelets were deposited in the liver sinusoids. In the present study, we investigated whether platelets are deposited in liver sinusoids in hepatic tumors in human and mice. CD61 immunohistochemical staining revealed the increased numbers of platelets were deposited in the sinusoids in foci, adenomas and hepatocellular carcinomas (HCC) induced by neonatal treatment with DEN compared to surrounding normal hepatic tissues. Moreover, increased numbers of platelets were deposited in sinusoids of dysplastic nodules and well-differentiated HCC compared to normal liver tissue and cirrhotic nodules in human. In addition, we evaluated the correlation between the tumor diameters and peripheral platelets numbers in the patients who were primarily diagnosed as HCC in our institute. The result indicated that the numbers of peripheral platelets are positively correlated with the tumor diameters. These results indicate that platelets contribute to development of HCC via interacting sinusoidal cells. Citation Format: Hiroki Tanaka, Kie Horioka, Masahiro Yamamoto, Koji Sawada, Takumu Hasebe, Kosuke Yamazaki, Katsuhiro Ogawa. Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5172.

The role of human CD46 in early xenoislet engraftment in a dual transplant model.

Membrane cofactor protein CD46 attenuates the complement cascade by facilitating cleavage of C3b and C4b. In solid organ xenotransplantation, organs expressing CD46 have been shown to resist hyperacute rejection. However, the incremental value of human CD46 expression for islet xenotransplantation remains poorly defined. This study attempted to delineate the role of CD46 in early neonatal porcine islet engraftment by comparing Gal-knocked out (GKO) and hCD46-transgenic (GKO/CD46) islets in a dual transplant model. Seven rhesus macaques underwent dual transplant and were sacrificed at 1hour (n=4) or 24hours (n=3). Both hemilivers were recovered and fixed for immunohistochemistry (CD46, insulin, neutrophil elastase, platelet, IgM, IgG, C3d, C4d, CD68, Caspase 3). Quantitative immunohistochemical analysis was performed using the Aperio Imagescope. Within 1hour of intraportal infusion of xenografts, no differences were observed between the two types of islets in terms of platelet, antibody, or complement deposition. Cellular infiltration and islet apoptotic activity were also similar at 1hour. At 24hours, GKO/CD46 islets demonstrated significantly less platelet deposition (P=0.01) and neutrophil infiltration (P=0.01) compared to GKO islets. In contrast, C3d (P=0.38) and C4d (P=0.45) deposition was equal between the two genotypes. Our findings suggest that expression of hCD46 on NPIs potentially provides a measurable incremental survival advantage in vivo by reducing early thrombo-inflammatory events associated with instant blood-mediated inflammatory reaction (IBMIR) following intraportal islet infusion.

In vitro investigation of chemical properties and biocompatibility of neurovascular braided implants

Braiding of Nitinol micro wires is an established technology for the manufacturing of fine-meshed neurovascular implants for tortuous vessel geometries. Electropolishing of wires before the braiding process has the potential to improve the in vitro behaviour in terms of thrombogenicity and endothelial cell proliferation. In this study, we present the first in vitro investigation of braided electropolished/blue oxide Nitinol samples in a blood flow loop, showing a significantly lower activation of the coagulation pathway (represented by the TAT III marker) and a tendency towards reduced platelet adhesion. Furthermore, we applied the same surface treatment on flat disks and measured protein adhesion as well as endothelial cell proliferation. We compared our results to non-electropolished samples with a native oxide surface. While platelet deposition was reduced on electropolished/blue oxide surface, a significant increase of endothelial cell seeding was observed. Investigation of inflammatory marker expression in endothelial cells provided divergent results depending on the marker tested, demanding closer investigation. Surface analysis using Auger electron spectroscopy revealed a thin layer mainly consisting of titanium oxynitride or titanium oxide + titanium nitride as a potential cause of the improved biological performance. Translated to the clinical field of intracranial aneurysm treatment, the improved biocompatibility has the potential to increase both safety (low thrombogenicity) and effectiveness (aneurysm neck reconstruction).

Evaluation of Microscopic Structure-Function Relationships of PEGylated Small Intestinal Submucosa Vascular Grafts for Arteriovenous Connection.

Vascular grafts are used as vascular access for hemodialysis, the most common renal replacement therapy to artificially clean blood waste after kidney malfunction. Despite that they are widely used in clinical practice, upon implantation, synthetic vasculars show complications such as thrombogenesis, reduced patency rates, low blood pressure, or even complete collapse. In this study, a C-shaped vascular graft was manufactured with small intestinal submucosa (SIS) and modified on the surface and the bulk of the material via conjugation of polyethylene glycol (PEG) to obtain a biocompatible and less thrombogenic vascular graft than the commercially available polytetrafluoroethylene (ePTFE) vascular grafts. Molecular weight and concentration of PEG molecules were systematically varied to gain insights into the underlying structure-function relationships. We analyzed the chemical, thermal, and mechanical properties of vascular grafts modified with 6 equiv of SIS-PEG 400 as well as cytotoxicity and in vitro platelet deposition. Immune response, patency rates, and extent of regeneration were also tested in vivo with the aid of swine animal models. Results showed that the conjugation levels achieved were sufficient to improve graft compliance, therefore approaching that of native vessels, while platelet deposition was altered leading to a 95% reduction compared with pristine SIS and 92% with respect to ePTFE. H&E staining on explanted samples corroborated SIS-PEG 400 biocompatibility and the ability to promote regeneration. The obtained results set solid foundations for the rational design and manufacture of a regenerative, small diameter vascular graft model and introduce an alternative to ePTFE vascular grafts for hemodialysis access.

Microfluidic and computational study of structural properties and resistance to flow of blood clots under arterial shear

The ability of a blood clot to modulate blood flow is determined by the clot’s resistance, which depends on its structural features. For a flow with arterial shear, we investigated the characteristic patterns relating to clot shape, size, and composition on the one hand, and its viscous resistance, intraclot axial flow velocity, and shear distributions on the other. We used microfluidic technology to measure the kinetics of platelet, thrombin, and fibrin accumulation at a thrombogenic surface coated with collagen and tissue factor (TF), the key clot-formation trigger. We subsequently utilized the obtained data to perform additional calibration and validation of a detailed computational fluid dynamics model of spatial clot growth under flow. We then ran model simulations to gain insights into the resistance of clots formed under our experimental conditions. We found that increased thrombogenic surface length and TF surface density enhanced the bulk thrombin and fibrin generation in a nonadditive, synergistic way. The height of the platelet deposition domain—and, therefore, clot occlusivity—was rather robust to thrombogenic surface length and TF density variations, but consistently increased with time. Clot viscous resistance was non-uniform and tended to be higher in the fibrin-rich, inner “core” region of the clot. Interestingly, despite intraclot structure and viscous resistance variations, intraclot flow velocity variations were minor compared to the abrupt decrease in flow velocity around the platelet deposition region. Our results shed new light on the connection between the structure of clots under arterial shear and spatiotemporal variations in their resistance to flow.

Disseminated Intravascular Coagulation in Cancer: An Update.

Cancer often leads to the activation of coagulation, manifesting as disseminated intravascular coagulation (DIC) in its most extreme form. DIC is characterized by systemic intravascular coagulation activation (leading to deposition of intravascular platelets and fibrin) and simultaneous consumption of coagulation proteins and thrombocytes (which may cause bleeding complications). The clinical course of DIC in patients with malignancies is typically less intense compared with DIC complicating alternative clinical settings, including systemic inflammatory responses following infection or traumatic injury. A more slowly proceeding, less fulminant, and widespread hemostatic derangement can remain asymptomatic. Eventually, the ongoing consumption may result in low levels of platelets and coagulation factors, and bleeding complications (frequently localized at the site of the tumor or distant metastases) may be the first clinical manifestation of DIC. An alternative clinical scenario is dominated by thrombotic complications, ranging from clinically manifest vascular thrombosis to microvascular platelet plugs. The main principle of DIC management is adequate treatment of the precipitating disorder; however, there are clinical presentations that may require additional supportive strategies specifically aimed at the amelioration of the coagulopathy.

Normothermic Machine Perfusion (NMP) Inhibits Proinflammatory Responses in the Liver and Promotes Regeneration.

Liver transplantation (LT) is a successful treatment for patients with liver failure. However, organ shortage results in over 11% of patients losing their chance of a transplant attributed to liver decompensation (LD) and death. Ischemia/reperfusion injury (IRI) following conventional cold storage (CS) is a major cause of injury leading to graft loss after LT. Normothermic machine perfusion (NMP), a method of organ preservation, provides oxygen and nutrition during preservation and allows aerobic metabolism. NMP has recently been shown to enable improved organ utilization and posttransplant outcomes following a phase I and a phase III randomized trial. The aim of the present study is to assess the impact of NMP on reducing IRI and to define the underlying mechanisms. We transplanted and compared 12 NMP with 27 CS-preserved livers by performing gene microarray, immunoprofiling of hepatic lymphocytes, and immunochemistry staining of liver tissues for assessing necrosis, platelet deposition, and neutrophil infiltration, and the status of steatosis after NMP or CS prereperfusion and postreperfusion. Recipients receiving NMP grafts showed significantly lower peak aspartate aminotransferase (AST) levels than those receiving CS grafts. NMP altered gene-expression profiles of liver tissue from proinflammation to prohealing and regeneration. NMP also reduced the number of interferon gamma (IFN-γ) and interleukin (IL)-17-producing T cells and enlarged the CD4pos CD25high CD127neg FOXP3pos regulatory T cell (Treg) pool. NMP liver tissues showed less necrosis and apoptosis in the parenchyma and fewer neutrophil infiltration compared to CS liver tissues. Conclusion: Reduced IRI in NMP recipients was the consequence of the combination of inhibiting inflammation and promoting graft regeneration.

Epidermal growth factor-like repeats of SCUBE1 derived from platelets are critical for thrombus formation.

SCUBE1 [signal peptide-CUB-epidermal growth factor (EGF) domain-containing protein 1], expressed in endothelial cells (ECs) and platelets, exists in soluble or membrane forms. We previously showed that soluble SCUBE1 is a biomarker for platelet activation and also an active participant of thrombosis. However, whether the adhesive module of its EGF-like repeats is essential and the specific contribution of SCUBE1 synthesized in ECs or platelets to thrombosis in vivo remain unclear. We generated new mutant (Δ2) mice lacking the entire EGF-like repeats to evaluate the module's functional importance during thrombogenesis in vivo. The Δ2 platelet-rich plasma showed markedly impaired platelet aggregation induced by agonists including adenosine diphosphate, collagen, the thrombin agonist PAR-4 peptide and the thromboxane A2 analogue U46619. Consistently, genetic ablation of the EGF-like repeats diminished arterial thrombosis and protected Δ2 mice against lethal thromboembolism. On flow chamber assay, whole blood isolated from Δ2 or wild-type (WT) mice pre-treated with blocking antibodies against the EGF-like repeats showed a significant decrease in platelet deposition and thrombus formation on collagen-coated surfaces under arterial shear rates. Moreover, we created animals expressing SCUBE1 only in ECs (S1-EC) or platelets (S1-PLT) by reciprocal bone-marrow transplantation between WT and Δ2 mice. The time of carotid arterial thrombosis induced by ferric chloride was normal in S1-PLT chimeric mice but much prolonged in S1-EC animals. We demonstrate that platelet-derived SCUBE1 plays a critical role in arterial thrombosis via its adhesive EGF-like repeats in vivo and suggest targeting these adhesive motifs of SCUBE1 for potential anti-thrombotic strategy.

Surface Modification of Electrospun Scaffolds for Endothelialization of Tissue-Engineered Vascular Grafts Using Human Cord Blood-Derived Endothelial Cells.

Tissue engineering has gained attention as an alternative approach for developing small diameter tissue-engineered vascular grafts intended for bypass surgery, as an option to treat coronary heart disease. To promote the formation of a healthy endothelial cell monolayer in the lumen of the graft, polycaprolactone/gelatin/fibrinogen scaffolds were developed, and the surface was modified using thermoforming and coating with collagen IV and fibronectin. Human cord blood-derived endothelial cells (hCB-ECs) were seeded onto the scaffolds and the important characteristics of a healthy endothelial cell layer were evaluated under static conditions using human umbilical vein endothelial cells as a control. We found that polycaprolactone/gelatin/fibrinogen scaffolds that were thermoformed and coated are the most suitable for endothelial cell growth. hCB-ECs can proliferate, produce endothelial nitric oxide synthase, respond to interleukin 1 beta, and reduce platelet deposition.

Evaluation of von Willebrand factor concentrates by platelet adhesion to collagen using an in vitro flow assay

BackgroundVon Willebrand disease (VWD) results from quantitative or qualitative deficiency of von Willebrand factor (VWF) and is treated using VWF‐containing concentrates. Several studies have compared the function of various VWF containing concentrates however this has not been performed using shear based assays. ObjectivesTo compare the platelet‐capture potential of 10 commercially available, plasma‐derived VWF concentrates under shear conditions. MethodsVWF containing concentrates were assessed for VWF:Ag, VWF:CB, VWF:RCo, factor VIII:C ADAMTS13 content, VWF multimeric profile and glycan content using lectin binding assays. Free‐thiol content of each concentrate was investigated using MPB binding assays. An in vitro flow assay was used to determine the ability of each concentrate to mediate platelet capture to collagen. ResultsVWF multimeric analysis revealed reduction of high molecular weight (HMW) forms in four of the concentrates (Alphante, Octanate and Haemoctin, and 8Y). The high MW multimer distribution of the remaining six concentrates (Optivate, Wilate, Fandhi, Wilfactin, Haemate P, and Voncento) was similar to the plasma control. Lectin analysis demonstrated that 8Y had increased amount of T‐antigen. Although platelet capture after 5 minutes perfusion was similar for all concentrates; Alphante, Octanate, and Haemoctin, demonstrated the lowest levels of platelet capture after 60 seconds of perfusion. Free‐thiol content and ADAMTS13 levels varied widely between the concentrates but was not correlated with function. ConclusionAlphanate, Octanate, and Haemoctin, lacked HMW multimers and had the lowest initial platelet capture levels suggesting that the presence of VWF HMW multimers are required for initial platelet deposition.

Tspan18 is a novel regulator of the Ca2+ channel Orai1 and von Willebrand factor release in endothelial cells.

Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific ‘partner proteins’ and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.

Antisense Oligonucleotide Targeting of Thrombopoietin Synthesis Reduces Platelet Count within the Hemostatic Range and Slows Progression of De Novo Mammary Carcinogenesis in the Mmtv-Pymt Mouse

Platelets have long been proposed to play a role in fostering neoplasias, ranging from tissue repair to cancer, but clinical evidence remains limited. Since available therapautics antagonizing hemostatic platelet functions lead to bleeding, the concept of targeting platelets in the context of cancer prevention or treatment has not been pursued in controlled clinical trials. To address this, we developed a novel antiplatelet strategy by partial inhibition of thrombopoiesis.We hypothesized that if platelets played a key role in fostering neoplastic malignancies, controlled platelet count reduction within a safe range would slow platelet-dependent cancer progression without adversely affecting hemostasis. Since the MPL ligand megakaryocyte growth and development factor (thrombopoietin, MGDF) is required for maintenance of normal platelet count, and the liver is responsible only for a portion of thrombopoietin synthesis, we tested this hypothesis by targeted knock-down of hepatic thrombopoietin synthesis using an antisense oligonucleotide (ASO) in mammary cancer-prone transgenic mice. We investigated whether partial reduction of thrombopoietin sythesis through ASO knock-down was hemostatically safe and impacted kinetics or penetrance of neoplastic progression in de novo mammary carcinogenesis in MMTV-PyMT mice.We designed and generated thrombopoietin ASO-s (TPO ASO) to achieve about 50% platelet count reduction in mice by inhibition of hepatic thrombopoietin gene expression. Then MMTV-PyMT transgenic mice were subcutaneously administered one of the effective TPO ASO-s, with treatment initiated at 40 days of age and before appearance of palpable (>2.0 mm) mammary tumors. We assessed the impact of saturating dose TPO ASO, including platelet count, plasma TPO level, and number of megakaryocytes in the femoral bone marrow. Mammary tumor growth, pulmonary metastases, and overall survival (fixed end-point study based on total tumor burden) of TPO ASO-treated mice were compared with untreated controls. At study endpoints, mammary carcinomas, platelet deposition, intra-tumoral vessel density, and Ki-67 positive cells were quantitatively evaluated.As anticipated from the drug screen, TPO ASO treatment at saturating effect reduced plasma thrombopoietin levels and blood platelet count by ~50% within 4 weeks, and the number of megakaryocytes in femoral bone marrow. TPO ASO also suppressed primary tumor growth (attached figure) and impaired development of pulmonary metastases (p<0.05), resulting in increased overall survival (p<0.001). Histological analysis of primary tumors revealed that TPO ASO significantly reduced mean platelet deposition in tumor vessels (from 54% to 26%; p<0.001), reduced vessel density (from 3.1% area to 1.5% area; p<0.05) and Ki-67 positive cells (from 62/field to 36/field; p<0.05) in primary tumors. Together, these results support the notion that strategies that safely reduce platelet presence or activity within developing mammary tumors, may limit neoplastic progression coincident with reduced angiogenesis.In summary, we found that TPO ASO reduces thrombopoietin levels and platelet count within the hemostatically safe range and significantly slows neoplastic progression of spontaneous mammary carcinomas in MMTV-PyMT mice. Overall, pharmacological knockdown of hepatic TPO synthesis is a new antiplatelet strategy that may be reasonably safe and effective in various platelet-driven disorders, including cancer. DisclosuresRevenko:Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights. Monia:Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights. Gruber:Aronora, Inc: Employment, Other: Intellectual property rights.

Computational Modeling and Microfluidics Reveal Characteristic Patterns of Regulation of Clot Structure and Mechanics By Tissue Factor Localization

The growth of a blood clot is initiated by tissue factor (TF) exposure and is expected to depend on TF localization (i.e., its level and spatial distribution). We sought to understand how the structural and mechanical properties of clots under flow are shaped by simultaneously varying the surface density of TF and its area of exposure. We used a microfluidic device harboring thrombogenic surfaces that differed in length and TF surface density. We perfused human whole blood through this device and continually measured platelet deposition, thrombin formation, and fibrin accumulation by means of fluorescence microscopy. Using our recently developed, detailed mathematical model of spatial clot growth under flow, we performed computational simulations to gain insights into the connection between the structure of a clot and its mechanical properties, such as resistance to blood flow and flow axial velocity.We detected a non-additive, synergistic influence of the thrombogenic surface length and TF surface density on bulk thrombin and fibrin generation. We found that thrombogenic surface length and TF density controlled not only bulk accumulation, but also the occlusivity of the deposited platelet mass, as well as clot resistance to flow. The extent of this control depended on the blood flow velocity. An increase in the TF surface density resulted in condition-dependent differential acceleration of platelet deposition, thrombin formation, and fibrin accumulation. The viscous resistance of the clot was characterized by spatial variations and was higher in the inner clot region. Notably, despite variations in the intraclot structure, variations in the intraclot flow velocity were minor compared to the abrupt decrease in flow velocity at the boundary of the platelet deposition domain.Our analysis revealed characteristic patterns that describe how the shape, size, internal structure, and viscous resistance of a clot depend on the surface density of TF and its area of exposure. Taken together, our results suggest that the structure and mechanics of a growing clot are correlated, but can differ in their regulation by the distinct aspects of TF localization. These findings provide new insights into how initiating signals can temporally and spatially affect thrombus growth under flow. DISCLAIMERThe opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the U.S. Army or of the U.S. Department of Defense. This abstract has been approved for public release with unlimited distribution. DisclosuresNo relevant conflicts of interest to declare.

The Gut Microbiota as an Influencing Factor of Arterial Thrombosis.

The mutualistic gut microbiota does not only impact the development and function of various immune cell types, but it also influences the function of the hepatic vascular endothelium and prothrombotic platelet function. With germ-free mouse models, we have demonstrated that gut-derived microbial-associated molecular patterns could stimulate hepatic von Willebrand factor (VWF) synthesis and plasmatic VWF levels through Toll-like receptor-2 (TLR2), thus defining the extent of platelet deposition to the subendothelial matrix of the ligation-injured common carotid artery. In addition to the microbiota-derived choline metabolite trimethylamine N-oxide and the microbiota's regulatory role on the colonic serotonin biosynthesis pathway, affecting prothrombotic platelet function, TLR2-regulated hepatic endothelial VWF synthesis and elevated VWF plasma levels constitute a pivotal mechanism of how the gut microbiota is linked to arterial thrombosis. Conceptually, in addition to the identified functions of the gut microbiota in modulating host nutrition and metabolism, our work places the innate immune functions of the liver sinusoidal endothelium as an actuating variable in arterial thrombus growth.

Active wrinkles to drive self-cleaning: A strategy for anti-thrombotic surfaces for vascular grafts

The inner surfaces of arteries and veins are naturally anti-thrombogenic, whereas synthetic materials placed in blood contact commonly experience thrombotic deposition that can lead to device failure or clinical complications. Presented here is a bioinspired strategy for self-cleaning anti-thrombotic surfaces using actuating surface topography. As a first test, wrinkled polydimethylsiloxane planar surfaces are constructed that can repeatedly transition between smooth and wrinkled states. When placed in contact with blood, these surfaces display markedly less platelet deposition than control samples. Second, for the specific application of prosthetic vascular grafts, the potential of using pulse pressure, i.e. the continual variation of blood pressure between systole and diastole, to drive topographic actuation was investigated. Soft cylindrical tubes with a luminal surface that transitioned between smooth and wrinkled states were constructed. Upon exposure to blood under continual pressure pulsation, these cylindrical tubes also showed reduced platelet deposition versus control samples under the same fluctuating pressure conditions. In both planar and cylindrical cases, significant reductions in thrombotic deposition were observed, even when the wrinkles had wavelengths of several tens of μm, far larger than individual platelets. We speculate that the observed thrombo-resistance behavior is attributable to a biofilm delamination process in which the bending energy within the biofilm overcomes interfacial adhesion. This novel strategy to reduce thrombotic deposition may be applicable to several types of medical devices placed into the circulatory system, particularly vascular grafts.