Surface Of Activated Platelets Research Articles

Biophysical Characterization of the Thermodynamic Basis and Binding Orientation for Factor VIII Membrane Association

After vesicular damage occurs, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated factor IX (fIX) on the surface of activated platelets during the blood coagulation cascade to form the intrinsic “tenase” complex. Once activated, fVIII dissociates from von Willebrand factor and C1 and C2 domains bind to activated platelet surfaces. Previous studies have identified that the C1 and C2 domains bind to negatively charged membranes through both surface exposed hydrophobic residues located in the beta hairpin loops and adjacent positively charged residues. The thermodynamics of fVIII lipid binding are currently uncharacterized and our current understanding of the binding orientation of the C1 and C2 domains of fVIII on lipid membranes is incomplete. In this study, we investigate the thermodynamics and binding orientation of the C domains via mutagenesis to both surface exposed hydrophobic residues and adjacent charged residues. Binding measurements between phosphatidylserine (PS) containing lipid nanodiscs and fVIII C domains have revealed that the C1 domain lipid binding is driven by enthalpic interactions, whereas the C2 domain binding is driven by entropic interactions. Binding experiments on fVIII C2 domain mutants for both surface exposed hydrophobic and positively charged residues have revealed differential disruption to lipid binding. Lastly, we have utilized molecular dynamics to perform simulations on the C1 and C2 domains binding to lipid nanodiscs to elucidate the binding orientation of the C domains and identify novel residues that interact with the membrane. Our results from these simulations agree with previous work, showing that C domain lipid binding centers on R2163 (C1) and R2320 (C2).

Cryo-EM Structure of Coagulation Factor VIII Bound to a Patient-Derived Anti-A2 Domain Antibody Inhibitor

Coagulation factor VIII (FVIII) promotes hemostasis by binding to activated factor IX on activated platelet surfaces to form the intrinsic tenase complex. The formation of inhibitory antibodies against FVIII occurs in approximately 30% of congenital hemophilia A patients receiving FVIII replacement therapy, significantly complicating treatments. By identifying the FVIII amino acids which bind to pathogenic inhibitory antibodies, FVIII replacement therapies can be engineered with reduced immunogenicity. Here, we aimed to structurally characterize FVIII bound to NB11B2, a patient-derived antibody inhibitor which binds to the A2 domain and disrupts tenase complex activity, using cryogenic electron microscopy (cryo-EM). The FVIII:NB11B2 structure was solved to a global resolution of 4.0 Å, representing the first structure of FVIII bound to an anti-A2 domain antibody inhibitor. Structural analysis revealed that NB11B2 binds to a discontinuous epitope encompassing FVIII residues S470-P472, R490-K493, and P502-E507. Furthermore, our structure identified a 20° swivel-like movement adopted by the C2 domain while the C1 domain remains immobile, consistent with a previous X-ray crystal structure of FVIII bound to an anti-C1 domain inhibitor. These results provide the structural basis for inhibition of FVIII by NB11B2 and elucidate a novel role for portions of the FVIII A2 domain in regulating tenase complex assembly and/or activity.

Factor VIII: A Dynamic Modulator of Hemostasis and Thrombosis in Trauma.

A trace amount of thrombin cleaves factor VIII (FVIII) into an active form (FVIIIa), which catalyzes FIXa-mediated activation of FX on the activated platelet surface. FVIII rapidly binds to von Willebrand factor (VWF) after secretion and becomes highly concentrated via VWF-platelet interaction at a site of endothelial inflammation or injury. Circulating levels of FVIII and VWF are influenced by age, blood type (nontype O > type O), and metabolic syndromes. In the latter, hypercoagulability is associated with chronic inflammation (known as thrombo-inflammation). In acute stress including trauma, releasable pools of FVIII/VWF are secreted from the Weibel-Palade bodies in the endothelium and then augment local platelet accumulation, thrombin generation, and leukocyte recruitment. Early systemic increases of FVIII/VWF (>200% of normal) levels in trauma result in a lower sensitivity of contact-activated clotting time (activated partial thromboplastin time [aPTT] or viscoelastic coagulation test [VCT]). However, in severely injured patients, multiple serine proteases (FXa plasmin and activated protein C [APC]) are locally activated and may be systemically released. Severity of traumatic injury correlates with prolonged aPTT and elevated activation markers of FXa, plasmin, and APC, culminating in a poor prognosis. In a subset of acute trauma patients, cryoprecipitate that contains fibrinogen, FVIII/VWF, and FXIII is theoretically advantageous over purified fibrinogen concentrate to promote stable clot formation, but comparative efficacy data are lacking. In chronic inflammation or subacute phase of trauma, elevated FVIII/VWF contributes to the pathogenesis of venous thrombosis by enhancing not only thrombin generation but also augmenting inflammatory functions. Future developments in coagulation monitoring specific to trauma patients, and targeted to enhancement or inhibition of FVIII/VWF, are likely to help clinicians gain better control of hemostasis and thromboprophylaxis. The main goal of this narrative is to review the physiological functions and regulations of FVIII and implications of FVIII in coagulation monitoring and thromboembolic complications in major trauma patients.

Humic Acids Inhibit Platelet Activation to Reduce Venous Thromboembolism in Mice.

We aimed to investigate the effects of the natural product humic acids (HA) on platelet activation and development of venous thromboembolism (VTE) in mice and further explore the relevant mechanism. Eight-week C57BL/6 mice were randomly assigned to three groups: sham operation group (n = 7), VTE group (n = 8), and VTE + HA group (n = 10). Thrombi were harvested to hematoxylin-eosin staining to evaluate the thrombolysis and recanalization of the thrombus. In addition, flow cytometry was performed to detect the expression levels of protein disulfide isomerase on endothelial-derived exosomes and glycoprotein IIb/IIIa on the surface of the activated platelets surface in plasma. Furthermore, the protein expression level of glycoprotein IIb/IIIa in thrombus was determined by immunohistochemistry and immunofluorescence. The length of thrombosis in the VTE + HA group was significantly shorter than that in the VTE group (P = 0.040). No significant differences were observed in thrombolysis and recanalization between the VTE + HA group and the VTE group (P > 0.05). The content of protein disulfide isomerase carried by endothelial-derived exosomes was significantly increased in the VTE group (P = 0.008) but significantly reduced by native humic acids (P = 0.012). Compared with the VTE group, the expression of glycoprotein IIb/IIIa on activated platelet surface in the VTE + HA group was significantly decreased (P = 0.002). The concentration of plasmatic P-selectin in the VTE group was significantly higher than that in the VTE + HA group (P < 0.001). We demonstrate that HA possess a pharmacological property that decreases venous thrombus formation in mice. The underlying mechanism is that HA could inhibit the expression of glycoprotein IIb/IIIa on the activated platelets surface by suppressing endothelial-derived exosomes carrying on protein disulfide isomerase, thereby blocking platelet activation.

Inhibition of platelet-surface-bound proteins during coagulation under flow I: TFPI

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Tissue factor pathway inhibitor (TFPI) is one such inhibitor, well known for its inhibitory action on the active enzyme complex comprising tissue factor (TF) and activated clotting factor VII. This complex forms when TF embedded in the blood vessel wall is exposed by injury and initiates coagulation. A different role for TFPI, independent of TF:VIIa, has recently been discovered whereby TFPI binds a partially cleaved form of clotting factor V (FV-h) and impedes thrombin generation on activated platelet surfaces. We hypothesized that this TF-independent inhibitory mechanism on platelet surfaces would be a more effective platform for TFPI than the TF-dependent one. We examined the effects of this mechanism on thrombin generation by including the relevant biochemical reactions into our previously validated mathematical model. Additionally, we included the ability of TFPI to bind directly to and inhibit platelet-bound FXa. The new model was sensitive to TFPI levels and, under some conditions, TFPI could completely shut down thrombin generation. This sensitivity was due entirely to the surface-mediated inhibitory reactions. The addition of the new TFPI reactions increased the threshold level of TF needed to elicit a strong thrombin response under flow, but the concentration of thrombin achieved, if there was a response, was unchanged. Interestingly, we found that direct binding of TFPI to platelet-bound FXa had a greater anticoagulant effect than did TFPI binding to FV-h alone, but that the greatest effects occurred if both reactions were at play. The model includes activated platelets’ release of FV species, and we explored the impact of varying the FV/FV-h composition of the releasate. We found that reducing the zymogen FV fraction of this pool, and thus increasing the fraction that is FV-h, led to acceleration of thrombin generation.

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.

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.

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

AbstractThe intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis–Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein–protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1–A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

SAXS Analysis of the Intrinsic Tenase Complex Bound to Lipid Nanodisc Highlights Intermolecular Contacts between Factors VIIIa/IXa

The intrinsic tenase (Xase) complex is formed by activated clotting factors VIII (fVIIIa) and IX (fIXa) on activated platelet surfaces and catalyzes the activation of factor X to promote blood coagulation. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering a mechanistic understanding of how mutations to fVIIIa or fIXa lead to thrombotic disorders. In the present study, we aimed to structurally characterize the Xase complex bound to a lipid nanodisc using biolayer interferometry and small-angle X-ray scattering. By immobilizing lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results suggest that interactions between the Gla and catalytic domains of fIXa and the C2 and A2 domains of fVIIIa, respectively, mediate Xase complex assembly. Additionally, our computational modelling highlighted a novel interaction between fIXa residue K80 and an acidic patch formed at the fVIIIa A1/A3 domain interface. Our model of the Xase complex sheds light on how hemophilia A/B-related mutations with varying severities disrupt Xase complex assembly. We also speculate on how gain-of-function mutations to fIXa residue R338, such as the Padua (R338L) and Shanghai (R338Q) variants, bind to the fVIIIa A2 domain and stabilize the Xase complex, leading to excessive blood clotting. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex. DisclosuresLollar: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

Intravenous Nanomedicine for Targeted Delivery of Thrombin to Augment Hemostasis

Non-compressible uncontrolled hemorrhage remains a major cause of mortality from traumatic injuries. Additionally, patients with congenital, disease-associated or drug-induced hemostatic dysfunctions, may often be at risk of excessive bleeding. Therefore, treatments that render rapid hemostasis are clinically significant in potentially saving lives. The clinical gold standard for this is the transfusion of whole blood (WB) or blood components (e.g. controlled ratios of platelets, RBCs, and plasma), as evidenced by several clinical studies (e.g. PROPPR, PROMMTT and PAMPer). However, the availability of such blood products is donor-dependent, their shelf-life is limited due to contamination risks, and, their portability and storage is often challenging. While extensive research efforts are currently being focused on addressing these challenges, e.g. using low titer Group O whole blood, cold-storage and freeze-drying of platelets and plasma, in vitro generation of platelets from iPSCs etc., a parallel research focus has emerged in designing biomaterials-based I.V.-administrable technologies (nanoparticles, polymers etc.) that can provide specific functional attributes of hemostasis while allowing donor-independent manufacturing, scale-up, and on-demand availability. Prominent examples of these are ‘synthetic platelet’ (SynthoPlate) nanoparticles that recapitulate platelet's binding interactions with von Willebrand Factor (vWF), collagen and active platelet integrin GPIIb-IIIa, flexible platelet-like particles (PLP) that bind fibrin to recapitulate platelet's biomechanical properties, fibrinogen function-mimicking nanoparticles that amplify the aggregation of active platelets, peptide-modified synthetic polymers (e.g. PolySTAT, HAPPI etc.) that render clot stabilization etc. In this framework, we present the design and evaluation of I.V.-administrable unique platelet-inspired nanoparticles that render injury site-targeted, enzyme-responsive direct delivery of thrombin, to site-specifically augment fibrin generation for hemostasis. Our design is inspired by platelets' crucial hemostatic mechanisms of : (i) rapidly accumulating at the injury site to form a plug and (ii) serving as a coagulation amplifier via presenting anionic phospholipids on the activated platelet surface to render tenase and prothrombinase factor assemblies leading to thrombin (FIIa) burst, which can then site-specifically convert fibrinogen to fibrin. Thrombin delivery to augment hemostasis is clinically well-accepted, as exemplified by products like Tisseel where thrombin and fibrinogen are co-delivered by syringe directly at wound site. Researchers have also studied thrombin-loaded topical dressings and topical administration of thrombin-loaded particles on wounds to mitigate bleeding, but these cannot be used intravenously. A recent interesting study has explored encapsulation of thrombin-loaded nanoparticles inside actual platelets with the idea of the particles being released (analogous to granule secretion) upon platelet activation, but this was only demonstrated in vitro because optimizing this complex strategy for consistent in vivo function may be challenging. Our approach circumvents these challenges by: (i) loading consistent amount of thrombin in I.V.-administrable lipid nanoparticles (LNPs), (ii) directly targeting the thrombin-loaded LNPs (TLNPs) to the injury site via specific binding to vWF and collagen, and (iii) releasing the loaded thrombin via particle destabilization by the action of injury site-specific enzyme phospholipase A2 for in situ fibrin production. We evaluated the TLNPs in vitro in human blood and plasma where hemostatic defects were created by platelet depletion and anticoagulant treatment. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM) based studies of clot characteristics and BioFlux microfluidics based real-time imaging of fibrin generation under simulated vascular flow conditions, confirmed the ability of TLNPs to restore fibrin generation in hemostatic dysfunction settings. Subsequently, the in vivo feasibility of these TLNPs was tested in a mouse tail-clip bleeding model where a combination of platelet depletion plus anticoagulant treatment was used to render significant hemostatic defect. TLNPs were able to effectively reduce tail-bleeding in mice. [Display omitted] DisclosuresSen Gupta: Haima Therapeutics: Other: Co-founder, Patents & Royalties: US 9107845, US 9107963.

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

AbstractThe αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Factor VIII and Factor V Membrane Bound Complexes.

The formation of membrane-bound complexes between specific coagulation factors at different cell surfaces is required for effective blood clotting. The most important of these complexes, the intrinsic Tenase and Prothrombinase complexes, are formed on the activated platelet surface during the propagation phase of coagulation. These two complexes are highly specific in their assembly mechanism and function modulated by anionic membranes, thus offering desirable targets for pharmaceutical interventions. Factor V (FV) and factor VIII (FVIII) are highly homologous non-enzymatic proteins. In their active state, FVa and FVIIIa serve as cofactors for the respective serine proteases factor Xa (FXa) and factor IXa (FIXa), significantly increasing their catalytic activity. This is achieved by forming well organized membrane-bound complexes at the phosphatidylserine rich activated platelet membrane in the presence of Ca2+ ions. The tenase (FVIIIa/FIXa) complex, catalyzes the proteolytic conversion of FX to FXa. Subsequently the prothrombinase (FVa/FXa) complex catalyzes the conversion of prothrombin to thrombin, required for efficient blood clotting. Although significant knowledge of FV and FVIII biochemistry and regulation has been achieved, the molecular mechanisms of their function are yet to be defined. Understanding the geometric assembly of the tenase and prothrombinase complexes is paramount in defining the structural basis of bleeding and thrombotic disorders. Such knowledge will enable the design of efficient pro- and anticoagulant therapies critical for regulating abnormal hemostasis. In this chapter, we will summarize the findings to date, showing our achievement in the field and outlining the future findings required to grasp the complexity of these proteins.

Activated factor VIII-mimicking effect by emicizumab on thrombus formation in type 2N von Willebrand disease under high shear flow conditions

IntroductionType 2N von Willebrand disease (2NVWD) is characterized by a mild to moderate reduction in plasma levels of factor (F)VIII associated with defective binding of von Willebrand factor (VWF) to FVIII and accelerated proteolysis and clearance of FVIII. The clinical phenotype in 2NVWD is often indistinguishable from mild/moderate hemophilia (H)A. Emicizumab is a bispecific antibody to FIX/FIXa and FX/FXa that mimics FVIIIa cofactor function, and emicizumab prophylaxis significantly reduces bleeding events in patients with severe HA. AimWe investigated the potential benefits of emicizumab in the hemostatic management of 2NVWD. Patients/methodsPerfusion chamber experiments were performed using whole blood from three 2NVWD patients with different clinical phenotypes (bleeding scores: 0, 6 and 20; mutations: p.R816W, p.R816W, and p.R365X/p.T791M, respectively). Furthermore, the impact of specific FVIII-VWF interactions on thrombus formation was investigated. ResultsDefective thrombus formation that correlated with bleeding phenotype was evident in these 2NVWD patients. Emicizumab improved surface coverage and thrombus height in all cases. Multi-color immunostaining of thrombi further demonstrated that emicizumab enhanced thrombin generation and fibrin formation. The addition of FVIII alone to 2NVWD whole blood did not augment thrombus formation, while supplementation with FVIII/VWF complex enhanced platelet-fibrin interactions. Furthermore, an anti-FVIII monoclonal antibody known to interrupt the release of FVIIIa from VWF depressed these effects. ConclusionsEmicizumab-induced enhancing effects of thrombus formation, independent on VWF, might be useful as an alternative therapy for 2NVWD patients. The extent of FVIII-VWF interaction should be optimal to deliver and release FVIII/FVIIIa on the activated platelet surface.

Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system

AbstractOur previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their procoagulant functions, platelets enhanced profibrinolytic potential by augmenting the accumulation of tissue-type plasminogen activator (tPA) and plasminogen, in vivo in a murine microthrombus model, and in vitro in a platelet-containing plasma clot model. To clarify the role of thrombin-activatable fibrinolysis inhibitor (TAFI), which regulates coagulation-dependent anti-fibrinolytic potential, we analyzed tPA-induced clot lysis times in platelet-containing plasma. Platelets prolonged clot lysis times in a concentration-dependent manner, which were successfully abolished by a thrombomodulin-neutralizing antibody or an activated TAFI inhibitor (TAFIaI). The results obtained using TAFI- or factor XIII–deficient plasma suggested that TAFI in plasma, but not in platelets, was essential for this prolongation, though its cross-linkage with fibrin was not necessary. Confocal laser scanning microscopy revealed that fluorescence-labeled plasminogen accumulated on activated platelet surfaces and propagated to the periphery, similar to the propagation of fibrinolysis. Plasminogen accumulation and propagation were both enhanced by TAFIaI, but only accumulation was enhanced by thrombomodulin-neutralizing antibody. Labeled TAFI also accumulated on both fibrin fibers and activated platelet surfaces, which were Lys-binding-site-dependent and Lys-binding-site-independent, respectively. Finally, TAFIaI significantly prolonged the occlusion times of tPA-containing whole blood in a microchip-based flow chamber system, suggesting that TAFI attenuated the tPA-dependent prolongation of clot formation under flow. Thus, activated platelet surfaces are targeted by plasma TAFI, to attenuate plasminogen accumulation and fibrinolysis, which may contribute to thrombogenicity under flow.

Procoagulant Phosphatidylserine-Exposing Platelets in vitro and in vivo.

The physiological heterogeneity of platelets leads to diverse responses and the formation of discrete subpopulations upon platelet stimulation. Procoagulant platelets are an example of such subpopulations, a key characteristic of which is exposure either of the anionic aminophospholipid phosphatidylserine (PS) or of tissue factor on the activated platelet surface. This review focuses on the former, in which PS exposure on a subpopulation of platelets facilitates assembly of the intrinsic tenase and prothrombinase complexes, thereby accelerating thrombin generation on the activated platelet surface, contributing importantly to the hemostatic process. Mechanisms involved in platelet PS exposure, and accompanying events, induced by physiologically relevant agonists are considered then contrasted with PS exposure resulting from intrinsic pathway-mediated apoptosis in platelets. Pathologies of PS exposure, both inherited and acquired, are described. A consideration of platelet PS exposure as an antithrombotic target concludes the review.

A mathematical model of coagulation under flow identifies factor V as a modifier of thrombin generation in hemophilia A

BackgroundThe variability in bleeding patterns among individuals with hemophilia A, who have similar factor VIII (FVIII) levels, is significant and the origins are unknown. ObjectiveTo use a previously validated mathematical model of flow‐mediated coagulation as a screening tool to identify parameters that are most likely to enhance thrombin generation in the context of FVIII deficiency. MethodsWe performed a global sensitivity analysis (GSA) on our mathematical model to identify potential modifiers of thrombin generation. Candidates from the GSA were confirmed by calibrated automated thrombography (CAT) and flow assays on collagen‐tissue factor (TF) surfaces at a shear rate of 100 per second. ResultsSimulations identified low‐normal factor V (FV) (50%) as the strongest modifier, with additional thrombin enhancement when combined with high‐normal prothrombin (150%). Low‐normal FV levels or partial FV inhibition (60% activity) augmented thrombin generation in FVIII‐inhibited or FVIII‐deficient plasma in CAT. Partial FV inhibition (60%) boosted fibrin deposition in flow assays performed with whole blood from individuals with mild and moderate FVIII deficiencies. These effects were amplified by high‐normal prothrombin levels in both experimental models. ConclusionsThese results show that low‐normal FV levels can enhance thrombin generation in hemophilia A. Further explorations with the mathematical model suggest a potential mechanism: lowering FV reduces competition between FV and FVIII for factor Xa (FXa) on activated platelet surfaces (APS), which enhances FVIII activation and rescues thrombin generation in FVIII‐deficient blood.

Contrast enhanced ultrasound molecular imaging of platelets in the inflammatory procession of atherosclerosis

Objective To assess the role of activated platelets in the inflammatory procession of atherosclerosis(AS) by ultrasound molecular imaging. Methods Sixty ApoE-/- mice were fed with high fat diet to establish AS model as experimental group, and 40 C57BL/6J mice were fed with normal diet as control group. Biotin-avidin bridging method was used to construct platelet-targeted microbubbles with recombinant vWF-A1 domain (Mb-A1), microbubbles carrying monoclonal antibodies to VCAM-1 (Mb-VCAM1) and microbubbles carrying IgG monoclonal antibody (Mb-ctrl). In vitro and in vivo experiments were carried out to evaluate the ability of Mb-A1 to target platelets on vascular endothelial surface. Contrast enhanced ultrasound molecular imaging of proximal ascending aorta was performed with Mb-A1, Mb-VCAM1 and Mb-ctrl. The expression and distribution of platelets and monocytes/macrophages on the endothelium of ascending aorta of AS mice were observed and analyzed by immunofluorescence staining. Results ①A large number of Mb-A1 adhering to the surface of activated platelets coated in Petri dishes were observed under fluoresce. ②After platelet immune-depletion in 30-week AS mice, the signal intensity of Mb-A1 decreased significantly in ascending aorta, while that of Mb-ctrl has no obvious change(P<0.05). ③In ApoE-/- mice, signals from platelet targeted microbubbles increased from 8 to 32 weeks of age in ApoE-/- mice, which coincided with the increase of signals from VCAM-1 targeted microbubbles(P<0.05). ④Activated platelets on the endothelial surface of ascending aorta increased progressively with age from 8 weeks, and partly overlapped with the distribution of monocytes/macrophages. Conclusions Platelets contribute to the initiation and progression of atherosclerosis as an inflammatory mediator through the interaction with vascular endothelium. Key words: Contrast enhanced ultrasound; Targeted microbubble; Atherosclerosis; Inflammation; Platelet

Activated Platelets‐Targeting Micelles with Controlled Drug Release for Effective Treatment of Primary and Metastatic Triple Negative Breast Cancer

AbstractThe frequent relapse and metastasis characteristics of triple negative breast cancer (TNBC) make it a fraught issue with very poor prognosis in clinic. An effective treatment for TNBC should prevent and even eliminate metastasis as well as suppress primary lesion expansion. Recent progress reveals that platelets can be recruited and activated by tumor cells through intercellular adhesion molecules (ICAM), and help aggressive circulating tumor cells (CTCs) form metastasis. Therefore, activated platelets are considered with possession of tumor‐homing, CTC‐capturing, and metastasis‐targeting abilities. In this work, a P‐selectin (expressed on activated platelet surface) targeting peptide (PSN) is modified on a redox‐responsive paclitaxel‐loaded micelle (PSN‐PEG‐SS‐PTX4 micelle) to utilize activated platelets as a “bridge” for interaction with cancer cells. The PSN‐modified micelle can easily adhere to the surface of activated platelets and subsequently capture CTCs in blood circulation. Compared to Taxol and PEG‐SS‐PTX4 micelle, PSN‐PEG‐SS‐PTX4 micelle also exhibits enhanced primary TNBC/metastasis targeting and penetrating effect through binding with tumor infiltrating platelets and thus significantly improves treatment outcome. More importantly, PSN‐PEG‐SS‐PTX4 micelle potently suppressed lung metastasis of TNBC and reduced incidence of distant liver metastasis. The activated platelet‐targeting redox‐responsive micelle system provides a promising prospect for the omnidirectional treatment of metastatic cancer.

The 3.2 Angstrom Crystal Structure of a Bioengineered Factor VIII Construct Illustrates Two Conformations of the C2 Domain

Treatment for hemophilia A consists of repeated intravenous infusions of blood coagulation factor VIII (fVIII), either recombinant or derived from pooled plasma cryoprecipitate. While several recombinant versions are clinically available, all existing fVIII replacement products express at low levels, harbor short half-lives, and are cost intensive to produce for prophylaxis and therapeutic intervention. Here, we report the 3.2 Å structure of a bioengineered construct of fVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with an improved expression profile and slower A2 domain dissociation following proteolytic activation of fVIII (Figure 1A). Previous structural knowledge of fVIII is at 3.7 Å resolution or lower, and these structures display inconsistencies in their atomic details. The structure reported herein shows remarkable conservation amongst the core folds and domain arrangement of the fVIII protein as well as provides a more detailed view of the interface between the A2 domain and the remaining fVIII structure. With two fVIII molecules in the asymmetric unit of the crystal, we independently observe two distinct conformations of the C2 domain relative to the remaining fVIII structure. While one model (chain A) superimposes with previously-determined fVIII structures without significant deviation, the second model (chain B) indicates a ‘barrel tilt and roll’ conformational change of the C2 domain that may have implications for how proteolytically activated fVIII binds to activated platelet surfaces (Figure 1B). The improved model and stereochemistry of the ET3i A chain was subsequently used as a scaffold to generate an improved, refined model of human fVIII. In the case of both original datasets to 3.7 Å (pdb#: 2R7E) and 3.98 Å (pdb#: 3CDZ), our new model based on the ET3i structure resulted in improved refinement statistics for human fVIII. These improved structures further illustrate the conserved nature of the ET3i protein as well as provide a more confident model of fVIII for next generation engineering efforts to develop fVIII therapeutics with longer half-lives, higher expression levels and lower immunogenicity. [Display omitted] DisclosuresDoering:Expression Therapeutics, LLC: Equity Ownership, Research Funding.

Pro- and anticoagulant factors facilitate thrombin generation and balance the haemostatic response to FEIBA(®) in prophylactic therapy.

FEIBA(®) consists of zymogens and traces of activated forms of procoagulant factors II, VII, IX, X, anticoagulants protein C and TFPI, and small amounts of cofactors FV, FVIII and protein S, in a balanced ratio. As shown previously, FII-FXa complex plays a key role in FEIBA's mode of action (MoA). Thrombin generation (TG) was measured by spiking coagulation factors, cofactors and inhibitors to high titer FVIII inhibitor plasma, and in plasma samples from patients in a phase 3 clinical study evaluating the safety and efficacy of FEIBA prophylaxis in haemophilia A patients with inhibitors. Increasing the FXa/FII ratio improved TG, while adding coagulation enzyme components had a negligible effect. Adding FX, FIX, and FVII increased the peak thrombin and decreased the lag time. The presence of FV and phospholipids led to faster TG, while protein C and protein S reduced the amount of peak thrombin. TFPI appeared to have no effect. Patients on prophylaxis with FEIBA(®) showed higher peak thrombin and AUC with elevated FII, FX, FIX, FVIIa, and protein C levels, and experienced significantly less bleeding episodes than those receiving on-demand treatment. These experiments showed that although the FII-FXa complex induced immediate thrombin formation on the activated platelet surface, other procoagulant components of FEIBA were necessary to achieve an optimal thrombin burst. The presence of the pro- and anti-coagulants in FEIBA provides a haemostatic balance, and is thus expected to prevent thrombotic events. Recent clinical data verified the postulated MoA of FEIBA in prophylaxis treatment.