Sites Of Vascular Damage Research Articles

Acute response of astrocytes under rose bengal photoinduction‐mediated vascular injury

AbstractBackgroundCerebrovascular pathology, often found in autopsy confirmed Alzheimer’s disease (AD), is widely believed to exacerbate AD pathophysiology and complicate anti‐AD treatment strategies. Perivascular cells such as astrocytes maintain the microenvironment for proper brain cell function and vascular integrity. Early occurrence of cerebral small vessel disease and astrocyte reactivity could lower the threshold of neuronal disorders and AD. Studies of astrocyte and microvascular pathology and function in the brain have been limited and reaction of astrocytes to vascular damage has yet to be fully understood.MethodAAV‐Gfa‐EGFP or AAV‐Gfa‐GCaMP6 was injected into the barrel cortex of wild type mice followed by glass window installation. After 3 weeks recovery, rhodamine dextran was IV‐injected and intravital brain imaging through two‐photon microscopy was performed at pre‐ and post‐photoinduction. Photoactivation of IV‐injected Rose Bengal dye (RB) technique was used to create vascular pathology. ImageJ was used for data analysis and GraphPad prism was used for statistical analysis.ResultAfter photoinduction, green fluorescence intensity was increased in blood stream of both EGFP and GCaMP expressing brains. This result indicates a leakage of brain parenchyma component to blood stream. Astrocytic EGFP fluorescence intensity and astrocyte processes complexity were reduced after photoinduction in a time‐dependent manner. Interestingly, astrocyte calcium signaling was strikingly increased after photoinduction and returned back to the baseline at ∼1,000s post‐induction. The highest peak of astrocyte calcium signaling was located near a site of vascular damage, indicated by an increased in rhodamine fluorescence intensity in the brain parenchyma area. Astrocytic GCaMP intensity at the astrocyte endfeet surrounding small vasculature was increased, on the other hand, GCaMP intensity at the astrocyte processes are decreased in a time‐dependent manner. Consistently, we observed a reduction of vascular diameter after photoinduction in both EGFP and GCaMP expressing brains.ConclusionAcute vascular injury induced by rose bengal photoinduction altered astrocyte integrity and cellular signaling. This injury model resembles vascular constriction and hemorrhages found in human brain that could accelerate neurodegeneration and AD progression.

Transient particle tracking microrheology of plasma coagulation via the intrinsic pathway

Abstract The maintenance of hemostasis to ensure vascular integrity is dependent upon the rapid conversion of zymogen species of the coagulation cascade to their enzymatically active forms. This process culminates in the generation of the serine protease thrombin and polymerization of fibrin to prevent vascular leak at sites of endothelial cell injury or loss of cellular junctions. Thrombin generation can be initiated by the extrinsic pathway of coagulation through exposure of blood to tissue factor at sites of vascular damage, or alternatively by the coagulation factor (F) XII activated by foreign surfaces with negative charges, such as glass, through the contact activation pathway. Here, we used transient particle tracking microrheology to investigate the mechanical properties of fibrin in response to thrombin generation downstream of both coagulation pathways. We found that the structural heterogeneity of fibrin formation was dependent on the reaction kinetics of thrombin generation. Pharmacological inhibition of FXII activity prolonged the time to form fibrin and increased the degree of heterogeneity of fibrin, resulting in fibrin clots with reduced mechanical properties. Taken together, this study demonstrates a dependency of the physical biology of fibrin formation on activation of the contact pathway of coagulation.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through β-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

A RARE CASE OF GLANZMANN THOMBASTHENIA

Background: Glanzmanns Thrombasthenia (GT) is a GPIIb/IIIa platelet surface receptor condition that is defined by a lack of platelet aggregation, either qualitatively or quantitatively. This receptors physiological function is to bind various sticky plasma proteins that aid platelet adhesion and aggregation, ensuring the development of thrombus at sites of vascular damage. Patient of GT typically presents with menorrhagia, easy bruising, epistaxis, and gum bleeding.Prolonged untreated or unsuccessfully treated hemorrhagia associated with GlanzmannsThrombasthenia may be life threatening. Case Description: A 12 years old girl presented with bleeding since 12 days with complaints of generalized weakness. The patient was then investigated to find the cause of puberty menorrhagia. Several blood investigations were performed, and a final diagnosis of Glanzmanns Thrombasthenia was made. Objective:To highlight a rare case of Glanzmanns Thrombasthenia with β-Thalassemia trait in a 12 years old girl with puberty menorrhagia.

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19.

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF–platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage.

Effect of pulsatility on shear-induced extensional behavior of Von Willebrand factor.

Patients with continuous flow ventricular assist devices (CF-VADs) are at high risk for non-surgical bleeding, speculated to associate with the loss of pulsatility following CF-VAD placement. It has been hypothesized that continuous shear stress causes elongation and increased enzymatic degradation of von Willebrand Factor (vWF), a key player in thrombus formation at sites of vascular damage. However, the role of loss of pulsatility on the unravelling behavior of vWF has not been widely explored. vWF molecules were immobilized on the surface of microfluidic devices and subjected to various pulsatile flow profiles, including continuous flow and pulsatile flow of different magnitudes, dQ/dt (i.e., first derivative of flow rate) of pulsatility and pulse frequencies to mimic in vivo shear flow environments with and without CF-VAD support. VWF elongation was observed using total internal reflection fluorescence (TIRF) microscopy. Besides, the vWF level is measured from the patients' blood sample before and after CF-VAD implantation from a clinical perspective. To our knowledge, this work is the first in providing direct, visual observation of single vWF molecule extension under controlled-pulsatile shear flow. Unravelling of vWF (total sample size n~200 molecules) is significantly reduced under pulsatile flow (p<0.01) compared to continuous flow. An increase in the magnitude of pulsatility further reduces unravelling lengths, while lower frequency of pulsatility (20 vs. 60 pulses per min) does not have a major effect on the maximum or minimum unravelling lengths. Evaluation of CF-VAD patient blood samples (n=13) demonstrates that vWF levels decreased by ~40% following CF-VAD placement (p<0.01), which correlates to single-molecule observations from a clinical point of view. Pulsatile flow reduces unfolding of vWF compared to continuous flow and a lower pulse frequency of 20 pulses/minute yielded comparable vWF unfolding to 60 pulses/minute. These findings could shed light on non-surgical bleeding associated with the loss of pulsatility following CF-VAD placement.

A clinical case of the successful use of recombinant activated VII coagulation factor in a newborn with sepsis

Recently, in the intensive care of acquired coagulopathies in children and newborns, synthetic coagulation factors have occupied an important place, with recombinant VII activated coagulation factor (rFVIIa) being used more often. It initiates hemostasis at the site of vascular damage, forms a complex with tissue factor, and ensures maximum platelet activation. The resulting complex stimulates the transition of blood coagulation factors IX and X to the active form IXa and Xa, then factor Xa leads to increased synthesis thrombin and the formation of a stable fibrin clot. Initially, rFVIIa was developed and used to treat bleeding and surgery in patients with hereditary or acquired hemophilia and a high titer of an inhibitor to coagulation factors VIII or IX. Currently, indications for its use have expanded significantly and it is effective in intensive therapy of other acquired coagulopathy, including the occurrence of coagulopathy due to sepsis. This article presents a clinical case of the successful use of rFVIIa in a newborn with sepsis and hemocogulation disorders. The description of each case is important for the accumulation of experience and the development of further algorithms for the treatment of newborns with sepsis, accompanied by impaired hemocoagulation, since there is currently no single effective management strategy for such patients.

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor.

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both "loss as well as gain of function" mutations observed in this domain. Naturally occurring "gain of function" mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These "gain of function" mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the "gain of function" effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.

Von Willebrand Factor for Aortic Valve Intervention: From Bench to Real-Time Bedside Assessment.

VWF (von Willebrand factor ) is a circulating multimeric blood glycoprotein. VWF plays a major role in primary hemostasis by promoting the adhesion of platelets to subendothelial collagen at sites of vascular damage and thereby promoting platelet aggregation. VWF is synthesized in endothelial cells and megakaryocytes. The VWF units dimerize and are transported into the Golgi apparatus, where disulfide bonds are formed leading to formation of VWF multimers. This large subunit combination is required to support its hemostatic function. VWF has the unique features to be circulating in an inactive coiled form, hiding binding domains for platelet receptors and subendothelial collagen. At the site of vascular injury, VWF binds to the exposed collagen and unfolds. Once VWF is unfolded, the VWF A1 domain is exposed allowing the binding of platelets via GP Ib (glycoprotein IB) receptor. After platelet activation, GP IIb/IIIa (glycoprotein IIb/IIIa) receptor becomes able to bind VWF C1 domain. The VWF conformation and activity is intimately related to shear conditions and blood flow. At high shear rate (beyond 10–15 pN), it becomes unfolded exposing binding sites but also the cleavage site in VWF A2 domain for ADAMTS13 (adisintegrin-like and metalloprotease thrombospondin) protease that conducts to its proteolysis. Overall, these environmental changes generate the modification of the conformation of VWF …

230 Tracking late outgrowth endothelial cells in an acute arterial injury model

AimLate outgrowth endothelial cells (EOC) are strong contenders to be the true circulating endothelial progenitor cells since they are capable of clonogenic expansion, exhibit a mature endothelial phenotype, and contribute...

Internalization of Tissue Factor-Rich Microvesicles by Platelets Occurs Independently of GPIIb-IIIa, and Involves CD36 Receptor, Serotonin Transporter and Cytoskeletal Assembly.

Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.

Probing ADAMTS13 substrate specificity using phage display.

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73.

New Structural Insights into High Affinity Membrane Binding By Coagulation Factor V/Va

Factor Va binds to membranes exposing phosphatidylserine (PS) with nanomolar affinity. This high affinity interaction plays a vital role in the assembly of membrane-bound prothrombinase thereby supporting robust thrombin formation at the site of vascular damage. Although the importance of the C1 and C2 domains of FVa in membrane binding has long been recognized, mechanistic details are incompletely understood. Unlike human prothrombinase, Pseudonaja textilis (common brown snake, P. tex) has evolved a membrane-independent form of prothrombinase composed of a FVa-like protein (VPtex) tightly bound to a FXa-like protein in solution. Our structural advances with FVPtex provide new tools to address major unresolved questions related to membrane binding by FVa. The high resolution (1.95Å) structure of VPtex resembles previously published structures of inactivated bovine FVa and human FVIII of lower resolution with all the structural features considered critical for membrane binding by FVa. However, VPtex bound to PS-containing membranes with very poor affinity (Kd > 1 µM). Substitution of 9 residues in the C1 and C2 domains of VPtex with residues present in the hemostatic form of FV present in the plasma of the snake yielded a derivative (VPtexC1C2) that bound to PS-containing membranes with nanomolar affinity equivalent to hFV. Interestingly, the newly acquired function of membrane binding in VPtexC1C2 does not affect its ability to function in solution, suggesting that membrane binding and solution-phase function are controlled independently. Variants containing substitutions in the individual C domains (VPtexC1 and VPtexC2) exhibited intermediate affinities (Kd=100 nM and Kd=50 nM) for binding to PS-containing membranes. However, the binding energy contributions from the individual C-domains did not additively explain the affinity of VPtexC1C2 for membranes. The large connection energy (-8.7 kcal/mole) implies substantial energetic expenditure, possibly through a conformational rearrangement, upon membrane binding. This correlates well with higher thermal factors observed in the C1 and C2 domains of structures of VPtexC1C2 and VPtexC2 as compared to VPtex. It is also supported by rapid kinetic studies illustrating equivalence in the bimolecular association rate constants for human Va and VPtex variants regardless of their membrane affinity. Thus, high affinity membrane binding results from large decreases in the dissociation rate constant expected from a conformational change that allows the protein to adopt a new stable membrane-bound configuration. A second explanation for the lack of an obvious correlation between x-ray structures of VPtexC1C2, VPtexC2 and VPtex and their affinity for membranes lies in the possibility that their solution-phase conformations differ. We explored this using small angle x-ray scattering (SAXS) of VPtex, VPtexC2 and VPtexC1C2 in solution. The low resolution SAXS envelope for VPtex could be accounted for by minor shifts in the individual domains, particularly in C1 and C2, as seen in the crystal structure. However, the SAXS envelopes for membrane binding variants (VPtexC2 and VPtexC1C2) showed major shape changes in the C-domains. Rigid body modeling revealed an increasingly extended end-on arrangement, rather than a side-by-side configuration, of the C1 and C2 domains seen in the x-ray structure and in the SAXS envelope for FVPtex. The C2-domain was found to extend away from the base of the C1 domain along the long axis of the molecule correlating major structural differences in these VPtex variants with increasing affinity for membranes. Accordingly, the spatial disposition of the C1 and C2 domains in VPtexC2 appears intermediate to their arrangement in VPtex and VPtexC1C2.These findings contrast to the arrangement seen in the crystal structures of all factor V forms, where the C-domains are arranged side-by-side, probably due to limitations imposed by crystal packing. Our SAXS studies provide clear evidence of an unforeseen framework of C-domains associated with the ability of factor V forms to bind to membranes with high affinity. The findings reveal new mechanistic insights into the structural correlates of the membrane binding function of factor V. DisclosuresCamire:Pfizer: Consultancy, Patents & Royalties, Research Funding.

Phosphodiesterase type 4 blockade prevents platelet-mediated neutrophil recruitment at the site of vascular injury.

Platelet-neutrophil interactions play a key role in cardiovascular disease and inflammatory processes. Src family kinases mediate P-selectin glycoprotein ligand-1-Mac-1 cross talk necessary for firm platelet-neutrophil adhesion. Because Src family kinase activity can be regulated by cAMP-dependent pathways, in this work, we evaluated the role of phosphodiesterases in the signaling events that are required to sustain platelet-neutrophil interactions and neutrophil recruitment at the site of vascular injury. In neutrophils exposed to P-selectin, selective phosphodiesterase 4 (PDE4) inhibition prevented Src family kinase-mediated phosphorylation of the proline-rich tyrosine kinase 2 on Tyr579/Tyr580. The effects of PDE4 inhibition required protein kinase A, likely through protein kinase A-mediated activation of COOH-terminal Src kinase, a major negative regulator of Src family kinases. PDE4, but not other phosphodiesterase inhibitors, reduced platelet-neutrophil conjugates as well as neutrophil firm adhesion on spread platelets under flow conditions. The effect of PDE4 inhibition on neutrophil adhesion was primarily mediated by downregulation of P-selectin-induced activation of Mac-1. In a murine model of endovascular injury, selective inhibition of PDE4 significantly reduced neutrophil recruitment at the site of vascular damage. This study identifies PDE4 as a central node in the signaling network that mediates platelet-neutrophil adhesion and suggests that pharmacological inhibition of PDE4 may represent a novel therapeutic avenue for the treatment of cardiovascular disease.

Regulation of Platelet Activity in a Changing Redox Environment

The regulation of platelet function is finely tuned by a balance between the vasculature's redox environment and the oxidative processes that occur in it. The activation of platelets at sites of vascular damage is essential for the maintenance of normal hemostasis. In the extracellular milieu, a normal redox environment is maintained by thiol/disulfide redox couples, which include reduced and oxidized glutathione (GSH/GSSG) and cysteine (Cys/CySS). Oxidative changes in either of the plasma redox potentials are directly linked with risk factors for cardiovascular disease. Many proteins found on the surface of platelets contain cysteine residues that are targets for oxidation. These include platelet-specific integrins and thiol isomerase enzymes that respond to changes in the extracellular redox environment, thus influencing normal platelet responses. The post-translational modification of critical cysteine thiol groups is linked to alterations in redox potentials and occurs both intracellularly and extracellularly in normal platelet activation. Platelet integrins, in particular, are prime targets for redox modification due to their high cysteine content. Although the role of thiol/disulfide bond exchange in platelet activation is established, the effects of a changing redox environment on platelet reactivity are unclear. A thorough understanding of these mechanisms and how they interact with other platelet signaling events is of the utmost importance for the development of novel therapeutic targets so that we can protect against inappropriate thrombus formation.

In Vivo Interrogation of a Human Von Willebrand Type A Domain.

Abstract 2176Proteins containing Von Willebrand A domains play critical roles in hemostasis and arterial thrombosis by promoting cell-cell and cell-ligand interactions. Central to these processes is the formation of an adhesive bond between the A1 domain containing protein Von Willebrand Factor (VWF) and its receptor on platelets known as GPIbα. Although ex-vivo approaches have broadened our understanding of the biophysical properties that govern the interaction between this human receptor–ligand pair, validation of hypotheses based on these intriguing studies will require an appropriate biological model that permits the study of such complex adhesive interactions under appropriate hemodynamic conditions; only then will it be possible to truly ascertain the biological relevance of in vitro observations as they pertain to hemostasis and thrombosis in humans. Moreover, a biological model that permits the study of the function of the human VWF-A1 domain in vivo will be of tremendous value in the development of drugs for use in disorders associated with thrombotic microangiopathy.Given the importance of the interaction between GPIbα and the A1 domain of VWF in hemostasis and thrombus formation in humans, we genetically modified the murine VWF genomic sequence so that it contains the majority of the human A1 domain (VWF HA1) in lieu of its murine counterpart. Mutant mice were viable, born in expected mendelian ratio, and had platelet counts comparable to WT littermates. Importantly, VWF gene transcription, antigen levels, and multimer pattern were also equivalent to WT controls. In contrast, hemostasis was significantly disrupted in homozygous VWF HA1 mice with majority of animals continuing to bleed for >10 minutes after removal of the distal tip of the tail. Evidence that the defect in hemostasis was due to the inability of murine platelets to accumulate at sites of vascular damage is demonstrated by a 4-fold reduction in thrombus size in response to laser-induced arteriolar injury in the microcirculation of the cremaster muscle. To further evaluate the ability of this modified VWF to support interactions with murine platelets, we surfaced immobilized plasma VWF from these animals onto a glass cover slip, which was then incorporated into a parallel plate flow system. WT mouse blood infused over the immobilized substrate was unable to support any significant interactions with plasma VWF HA1 (wall shear rate of 1,600s−1). By contrast, human platelets rapidly accumulated on surface-immobilized VWF HA1 at levels observed for its human plasma counterpart; human platelets administered to VWF HA1 mice were also capable of restoring hemostasis in these animals and formed occlusive thrombi in response to laser-induced arterial injury. Remarkably, the human A1 domain contained within murine VWF was able to support ristocetin-induced human platelet agglutination whereas wild-type murine plasma VWF did not. We are now taking the next important steps in defining the in vivo consequences of altered bond formation between the human VWF-A1 domain and GPIbα as well as evaluating the anti-thrombotic effects of compounds designed to disrupt this interaction in patients with disease-associated thrombotic microangiopathy. Disclosures:No relevant conflicts of interest to declare.

Von Willebrand Factor

Thrombus formation is of paramount importance in the pathophysiology of acute ischemic stroke. Current antithrombotics used to treat or prevent cerebral ischemia are only moderately effective or bear an increased risk of severe bleeding. von Willebrand factor (VWF) has long been known to be a key player in thrombus formation at sites of vascular damage. While the association between VWF and coronary heart disease has been well studied, knowledge about the role of VWF in stroke is much more limited. However, in recent years, an increasing amount of clinical and preclinical evidence has revealed the critical involvement of VWF in stroke development. This review summarizes the latest insights into the pathophysiologic role of VWF-related processes in ischemic brain injury under experimental conditions and in humans. Potential clinical merits of novel inhibitors of VWF-mediated platelet adhesion and activation as powerful and safe tools to combat thromboembolic disorders including ischemic stroke are discussed. Preclinical and clinical evidence illustrates an important role of VWF in ischemic stroke, suggesting that VWF could become a promising target in stroke therapy.

Excess Bleeding Risk in Patients Taking SSRIs plus Antiplatelet Drugs

Release of serotonin by platelets enhances platelet aggregation at sites of vascular damage. By depleting platelets of serotonin, selective serotonin

The Functional Role of TLR9 in Human Platelets

Abstract 366Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. This is perhaps best exemplified by the observation that severe thrombocytopenia is associated with sepsis. While recent phenotypic and functional studies have addressed the roles of TLRs 2 and 4 in human/murine PLTs, which are primarily extracellular receptors, very little is known about the cellular localization, expression, and physiological significance of intracellular TLR9. Here we show that TLR9 localizes to unique granular compartments along the cell periphery (underlying the plasma membrane) in human PLTs. While thought to reside exclusively in the endosomes of monocytes, macrophages, and plasmacytoid dendritic cells, TLR9 expression is significantly enhanced in PLTs following incubation with thombin, ADP, PMA, CRP and type IV collagen, suggesting activation-mediated granule release. Surprisingly, TLR9 surface expression did not coincide with CD62P and CD61 expression levels upon PLT activation (which were not increased following type IV collagen incubation), and TLR9 was shown not to co-localize with known alpha-granule (fibrinogen, CD62P, PDGF-B, VEGF, CD42a, CD42b), dense granule (serotonin), endosomal (M6P, syntaxin-13), or lysosomal (LAMP-1) proteins. Immunogold electron microscopy revealed that TLR9 was expressed instead in a novel intracellular electron-dense granule (T-granule) that is mostly distributed along the plasma membrane. Incubation of resting PLTs with synthetic unmethylated Type C CpG dinucleotides (characteristic of bacterial/viral DNA) resulted in increased TLR9 surface expression, followed by increased Type C CpG sequestration and CD62P surface expression over 20 minutes. Interestingly, when TLR9 surface expression was specifically upregulated by pre-incubating PLTs with type IV collagen, subsequent incubation with Type C CpG resulted in considerably increased Type C CpG sequestration, CD62P surface expression, and PLT aggregation within 30 seconds of addition. These results imply that PLTs must be primed to express TLR9 on their surface prior to signal transduction through TLR9, and provide a mechanism for PLT regulation of the immune response to infection in human whole blood by sequestering bacterial/viral DNA and marking themselves for clearance. Taken together, this paper; (1) tracks TLR9 to a new intracellular compartment in PLTs, (2) describes a novel mechanism of TLR9 signaling, and (3) reveals a unique role for human PLTs as mediators of innate immunity at sites of vascular damage. Disclosures:No relevant conflicts of interest to declare.

Tissue Factor Is Internalized by Platelets Through Activation of RhoA and PI3-Kinase Promoting Reversible Cytoskeletal Assembly,

Abstract 3264TF associated with platelets may play a critical role in the development of ischemic complications at sites of vascular damage. Platelets can internalize and store tissue factor-rich microvesicles from human origin (hTF), a process that can be potentiated by serotonin (5-HT) (Galan et al, Thromb Haemost. 102:511, 2009). There is limited knowledge on the mechanisms involved in the uptake, redistribution and functional expression of TF by platelets. We have investigated the mechanisms implied in the uptake and traffic of hTF with especial attention to CD36 and the 5HT transporter receptor (SERT), but also to the involvement of PI3-kinase, small GTPases (RhoA) and modifications of cytoskeletal organization during vesicular trafficking. Isolated platelets were exposed to hTF for up to 10 min. Cytoskeletal assembly and activation of RhoA were analyzed by electrophoresis and ELISA, respectively. Aggregometry and ultrastructural microscopy were used to assess platelet activation and hTF uptake. Protein arrays were used to evaluate changes in signaling molecules induced by hTF. In addition, inhibitory strategies to block GPIIbIIIa (Reopro), the scavenger receptor GPIV (anti-CD36), the SERT (S-Citalopram, SCit), and PI3-kinase (Wortmannin, Wo) were applied to platelets prior exposure to hTF. Internalization of hTF resulted in reversible actin polymerization and association of contractile proteins (alpha-actinin, actin binding protein and myosin) to the cytoskeleton, being maximal at 1 min. Maximal activation of RhoA occurred at 1 min, decreasing gradually afterwards, while activation of PI3-kinase occurred later (at 5 min). Screening of signaling proteins revealed activation of Akt, ERK1/2, and SNAP-25, among others. All inhibitory strategies fully prevented platelet aggregation to hTF. Uptake of hTF was slightly prevented by Reopro, while both anti-CD36 and Wo partially inhibited hTF uptake and, interestingly, SCit was the most efficient. We conclude that uptake of hTF by platelets promotes activation of specific signaling molecules with reversible cytoskeletal assembly and membrane fusion. While it may be difficult to design specific strategies to prevent the activation of small GTPases, cytoskeletal arrangements or membrane fusion specifically for platelets, our results indicate that the CD36 scavenger receptor and the 5-HT transporter (SERT) may become realistic therapeutic targets to prevent or reduce the association of TF to platelets and hence its possible prothrombotic action.Grants: SAF2009-10365, Red HERACLES RD06/0009/1003 Disclosures:No relevant conflicts of interest to declare.