Genetically encoded biocompatible anti-coagulant protein-coated coronary artery stents drive endothelialization.

In response to the critical demand for advancements in coronary artery stents, this study addresses the challenges associated with arterial recoil and restenosis post-angioplasty and the imperative to encourage rapid re-endothelialization for minimizing thrombosis risks. We employed an innovative approach inspired by mussel adhesion, incorporating placental anticoagulant protein (AnnexinV) on stent design. The introduction of a post-translationally modified catecholic amino acid L-3,4-dihydroxyphenylalanine (L-Dopa), mimicking mussel characteristics, allowed for effective surface modification of Stainless steel stents through genetic code engineering in AnnexinV (AnxDopa). The efficacy of AnxDopa was analyzed through microscale thermophoresis and flow cytometry, confirming AnxDopa's exceptional binding with phosphatidylserine and activated platelets. AnxDopa coated stainless steel demonstrates remarkable bio-, hemo-, and immuno-compatibility, preventing smooth muscle cell proliferation, platelet adhesion, and fibrin formation. It acts as an interface between the stent and biological fluid, which facilitates the anticoagulation and rapid endothelialization. Surface modification of SS verified through XPS analysis and contact angle measurement attests to the efficacy of AnxDopa mediated surface modification. The hydrophilic nature of the AnxDopa-coated surface enhanced the endothelialization through increased protein absorption. This approach represents a significant stride in developing coronary stents with improved biocompatibility and reduced restenosis risks, offering valuable contributions to scientific and clinical realms alike.

In vivo assessment of dual-function submicron textured nitric oxide releasing catheters in a 7-day rabbit model.

Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. Herein, a dualfunction submicron textured nitric oxide (NO)-releasing catheter was developed. The hemocompatibility and antithrombotic activity of vascular catheters were evaluated in both 20 h in vitro blood loop and 7 d in vivo rabbit model. Surface characterization assessments via atomic force microscopy show the durability of the submicron pattern after incorporation of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The SNAP-doped catheters exhibited prolonged and controlled NO release mimicking the levels released by endothelium. Fabricated catheters showed cytocompatibility when evaluated against BJ human fibroblast cell lines. After 20h in vitro evaluation of catheters in a blood loop, textured-NO catheters exhibited a 13-times reduction in surface thrombus formation compared to the control catheters, which had 83% of the total area covered by clots. After the 7 d in vivo rabbit model, analysis on the catheter surface was examined via scanning electron microscopy, where significant reduction of platelet adhesion, fibrin mesh, and thrombi can be observed on the NO-releasing textured surfaces. Moreover, compared to relative controls, a 63% reduction in the degree of thrombus formation within the jugular vein was observed. Decreased levels of fibrotic tissue decomposition on the jugular vein and reduced platelet adhesion and thrombus formation on the texture of the NO-releasing catheter surface are indications of mitigated foreign body response. This study demonstrated a biocompatible and robust dual-functioning textured NO PU catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. STATEMENT OF SIGNIFICANCE: Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. This study demonstrated a robust, biocompatible, dual-functioning textured nitric oxide (NO) polyurethane catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. The fabricated catheters exhibited prolonged and controlled NO release that mimics endothelium levels. After the 7 d in vivo model, a significant reduction in platelet adhesion, fibrin mesh, and thrombi was observed on the NO-releasing textured catheters, along with decreased levels of fibrotic tissue decomposition on the jugular vein. Results illustrate that NO-textured catheter surface mitigates foreign body response.

Intelligent Design of Antithrombotic Peptide Targeting Collagen.

Binding of blood components to collagen was proved to be a key step in thrombus formation. Intelligent Design of Protein Matcher (IDProMat), a neural network model, was then developed based on the principle of seq2seq to design an antithrombotic peptide targeting collagen. The encoding and decoding of peptide sequence data and the interaction patterns of peptide chains at the interface were studied, and then, IDProMat was applied to the design of peptides to cover collagen. The 99.3% decrease in seq2seq loss and 58.3% decrease in MLP loss demonstrated that IDProMat learned the interaction patterns between residues at the binding interface. An efficient peptide, LRWNSYY, was then designed using this model. Validations on its binding on collagen and its inhibition of platelet adhesion were obtained using docking, MD simulations, and experimental approaches.

Ethyl-acetate extract of Spatholobi Caulis blocked the pro-metastatic support from the hemato-microenvironment of colon cancer by specific disruption of tumor-platelet adhesion.

BACKGROUND: Within the pro-metastatic hemato-microenvironment, interaction between platelets and tumor cells provides essential support for tumor cells by inducing Epithelial-Mesenchymal Transition (EMT), which greatly increases the stemness of colon cancer cells. Pharmacologically, although platelet deactivation has proved to be benefit against metastasis, its wide application is severely restricted due to the bleeding risk. Spatholobi Caulis, a traditional Chinese herb with circulatory promotion and blood stasis removal activity, has been proved to be clinically effective in malignant medication, leaving its mechanistic relevance to tumor-platelet interaction largely unknown. METHODS: Firstly, MC38-Luc cells were injected into tail-vein in C57BL/6 mice to establish hematogenous metastasis model and the anti-metastasis effects of SEA were evaluated by using a small-animal imaging system. Then, we evaluated the anti-tumor-platelet interaction efficacy of SEA using a tumor-specific induced platelet aggregation model. Platelet aggregation was specifically induced by tumor cells in vitro. Furthermore, to clarify the anti-metastatic effects of SEA is mainly attributed to its blockage on tumor-platelet interaction, after co-culture with tumor cells and platelets (with or without SEA), MC38-Luc cells were injected into the tail-vein and finally count the total of photons quantitatively. Besides, to clarify the blocking pattern of SEA within the tumor-platelet complex, the dependence of SEA on different fractions from activated platelets was tested. Lastly, molecular docking screening were performed to screen potential effective compounds and we used ß-catenin blockers to verify the pathways involved in SEA blocking tumor-platelet interaction. RESULTS: Our study showed that SEA was effective in blocking tumor-platelet specific interaction: (1) Through CCK-8 and LDH assays, SEA showed no cytotoxic effects on tumor cells and platelets. On this basis, by the tail vein injection model, the photon counts in the SEA group was significantly lower than model group, indicating that SEA effectively reduced metastasis. (2) In the "tumor-platelet" co-culture model, SEA effectively inhibited the progression of EMT and cancer stemness signatures of MC38 cells in the model group. (3) In mechanism study, by using the specific inhibitors for galectin-3 (GB1107) andWNT (IWR) respectively, we proved that SEA inhibits the activation of the galectin-3-mediated ß-catenin activation. CONCLUSION: By highlighting the pro-metastatic effects of galectin-3-mediated tumor-platelet adhesion, our study provided indicative evidence for Spatholobi Caulis as the representative candidate for anti-metastatic therapy.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module.

ABSTRACT: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear.

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s-1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

Chronic edible dosing of Δ9-tetrahydrocannabinol (THC) in nonhuman primates reduces systemic platelet activity and function.

Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.

Janus kinase inhibitors ruxolitinib and baricitinib impair glycoprotein-VI mediated platelet function.

Several Janus kinase (JAK) inhibitors (jakinibs) have recently been approved to treat inflammatory, autoimmune and hematological conditions. Despite emerging roles for JAKs and downstream signal transducer and activator of transcription (STAT) proteins in platelets, it remains unknown whether jakinibs affect platelet function. Here, we profile platelet biochemical and physiological responses in vitro in the presence of five different clinically relevant jakinibs, including ruxolitinib, upadacitinib, oclacitinib, baricitinib and tofacitinib. Flow cytometry, microscopy and other assays found that potent JAK1/2 inhibitors baricitinib and ruxolitinib reduced platelet adhesion to collagen, as well as platelet aggregation, secretion and integrin αIIbß3 activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP-XL). Western blot analysis demonstrated that jakinibs reduced Akt phosphorylation and activation following GPVI activation, where ruxolitinib and baricitinib prevented DAPP1 phosphorylation. In contrast, jakinibs had no effects on platelet responses to thrombin. Inhibitors of GPVI and JAK signaling also abrogated platelet STAT5 phosphorylation following CRP-XL stimulation. Additional pharmacologic experiments supported roles for STAT5 in platelet secretion, integrin activation and cytoskeletal responses. Together, our results demonstrate that ruxolitinib and baricitinib have inhibitory effects on platelet function in vitro and support roles for JAK/STAT5 pathways in GPVI/ITAM mediated platelet function.

Vascular Polyurethane Prostheses Modified with a Bioactive Coating-Physicochemical, Mechanical and Biological Properties.

This study describes a method for the modification of polyurethane small-diameter (5 mm) vascular prostheses obtained with the phase inversion method. The modification process involves two steps: the introduction of a linker (acrylic acid) and a peptide (REDV and YIGSR). FTIR and XPS analysis confirmed the process of chemical modification. The obtained prostheses had a porosity of approx. 60%, Young's Modulus in the range of 9-11 MPa, and a water contact angle around 40°. Endothelial (EC) and smooth muscle (SMC) cell co-culture showed that the surfaces modified with peptides increase the adhesion of ECs. At the same time, SMCs adhesion was low both on unmodified and peptide-modified surfaces. Analysis of blood-materials interaction showed high hemocompatibility of obtained materials. The whole blood clotting time assay showed differences in the amount of free hemoglobin present in blood contacted with different materials. It can be concluded that the peptide coating increased the hemocompatibility of the surface by increasing ECs adhesion and, at the same time, decreasing platelet adhesion. When comparing both types of peptide coatings, more promising results were obtained for the surfaces coated with the YISGR than REDV-coated prostheses.

DMAG, a novel countermeasure for the treatment of thrombocytopenia.

BACKGROUND: Thrombocytopenia is one of the most common hematological disease that can be life-threatening caused by bleeding complications. However, the treatment options for thrombocytopenia remain limited. METHODS: In this study, giemsa staining, phalloidin staining, immunofluorescence and flow cytometry were used to identify the effects of 3,3'-di-O-methylellagic acid 4'-glucoside (DMAG), a natural ellagic acid derived from Sanguisorba officinalis L. (SOL) on megakaryocyte differentiation in HEL cells. Then, thrombocytopenia mice model was constructed by X-ray irradiation to evaluate the therapeutic action of DMAG on thrombocytopenia. Furthermore, the effects of DMAG on platelet function were evaluated by tail bleeding time, platelet aggregation and platelet adhesion assays. Next, network pharmacology approaches were carried out to identify the targets of DMAG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to elucidate the underling mechanism of DMAG against thrombocytopenia. Finally, molecular docking simulation, molecular dynamics simulation and western blot analysis were used to explore the relationship between DAMG with its targets. RESULTS: DMAG significantly promoted megakaryocyte differentiation of HEL cells. DMAG administration accelerated platelet recovery and megakaryopoiesis, shortened tail bleeding time, strengthened platelet aggregation and adhesion in thrombocytopenia mice. Network pharmacology revealed that ITGA2B, ITGB3, VWF, PLEK, TLR2, BCL2, BCL2L1 and TNF were the core targets of DMAG. GO and KEGG pathway enrichment analyses suggested that the core targets of DMAG were enriched in PI3K-Akt signaling pathway, hematopoietic cell lineage, ECM-receptor interaction and platelet activation. Molecular docking simulation and molecular dynamics simulation further indicated that ITGA2B, ITGB3, PLEK and TLR2 displayed strong binding ability with DMAG. Finally, western blot analysis evidenced that DMAG up-regulated the expression of ITGA2B, ITGB3, VWF, p-Akt and PLEK. CONCLUSION: DMAG plays a critical role in promoting megakaryocytes differentiation and platelets production and might be a promising medicine for the treatment of thrombocytopenia.

Preparations from selected cucurbit vegetables as antiplatelet agents.

Increased blood platelet activation plays an important role in cardiovascular diseases (CVDs). Recent experiments indicate that certain fruits and vegetables, including onion, garlic, and beetroot, have anti-platelet potential and therefore may reduce the likelihood of CVDs. While vegetables from the Cucuritaceae family are known to exerting beneficial antioxidant and anti-inflammatory effects, their effects on blood platelet activation are poorly understood. Therefore, the aim of the present study was to determine the effect on platelet adhesion of preparations from selected cucurbits: pumpkin (Cucurbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds). It also evaluates the activity of these preparations on enzymatic lipid peroxidation in thrombin-activated washed blood platelets by TBARS assay. The study also determines the anti-platelet properties of these five cucurbit preparations in whole blood by flow cytometry and with the total thrombus-formation analysis system (T-TAS) and evaluates the cytotoxicity of the tested preparations against platelets based on LDH activity. The results indicate that the yellow Cucurbita pepo var. patisoniana preparation demonstrated stronger anti-platelet properties than the other tested preparations, reducing the adhesion of thrombin-activated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. None of the preparations was found to cause platelet lysis. Our findings provide new information on the anti-platelet activity of the tested cucurbit preparations and their potential for treating CVDs associated with platelet hyperactivity.

The Toll-Like Receptor 2 Ligand Pam2CSK4 Activates Platelet Nuclear Factor-κB and Bruton's Tyrosine Kinase Signaling to Promote Platelet-Endothelial Cell Interactions.

Circulating platelets establish a variety of immunological programs and orchestrate inflammatory responses at the endothelium. Platelets express the innate immunity family of Toll-like receptors (TLRs). While TLR2/TLR1 ligands are known to activate platelets, the effects of TLR2/TLR6 ligands on platelet function remain unclear. Here, we aim to determine whether the TLR2/TLR6 agonists Pam2CSK4 and FSL-1 activate human platelets. In addition, human umbilical vein endothelial cells (HUVECs) and platelets were co-cultured to analyze the role of platelet TLR2/TLR6 on inflammation and adhesion to endothelial cells. Pam2CSK4, but not FSL-1, induced platelet granule secretion and integrin αIIbß3 activation in a concentration-dependent manner. Moreover, Pam2CSK4 promoted platelet aggregation and increased platelet adhesion to collagen-coated surfaces. Mechanistic studies with blocking antibodies and pharmacologic inhibitors demonstrated that the TLR2/Nuclear factor-κB axis, Bruton's-tyrosine kinase, and a secondary ADP feedback loop are involved in Pam2CSK4-induced platelet functional responses. Interestingly, Pam2CSK4 showed cooperation with immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling to enhance platelet activation. Finally, the presence of platelets increased inflammatory responses in HUVECs treated with Pam2CSK4, and platelets challenged with Pam2CSK4 showed increased adhesion to HUVECs under static and physiologically relevant flow conditions. Herein, we define a functional role for platelet TLR2-mediated signaling, which may represent a druggable target to dampen excessive platelet activation in thrombo-inflammatory diseases.

Dressing Blood-Contacting Materials by a Stable Hydrogel Coating with Embedded Antimicrobial Peptides for Robust Antibacterial and Antithrombus Properties.

Although dressing blood-contacting devices with robust and synergistic antibacterial and antithrombus properties has been explored for several decades, it still remains a great challenge. In order to endow materials with remarkable antibacterial and antithrombus abilities, a stable and antifouling hydrogel coating was developed via surface-initiated polymerization of sulfobetaine methacrylate and acrylic acid on a polymeric substrate followed by embedding of antimicrobial peptides (AMPs), including WR (sequence: WRWRWR-NH2) or Bac2A (sequence: RLARIVVIRVAR-NH2) AMPs. The chemical composition of the AMP-embedded hydrogel coating was determined through XPS, zeta potential, and SEM-EDS measurements. The AMP-embedded antifouling hydrogel coating showed not only good hemocompatibility but also excellent bactericidal and antiadhesion properties against Gram-positive and Gram-negative bacteria. Moreover, the hydrogel coating could protect the AMPs with long-term bioactivity and cover the positive charge of the dotted distributed AMPs, which in turn well retained the hemocompatibility and antifouling capacity of the bulk hydrogels. Furthermore, the microbiological results of animal experiments also verified the anti-infection performance in vivo. Histological and immunological data further indicated that the hydrogel coating had an excellent anti-inflammatory function. Therefore, the present study might provide a promising approach to prevent bacterial infections and thrombosis in clinical applications of blood-contacting devices and related implants.

Inhibition of Phosphodiesterase 3A by Cilostazol Dampens Proinflammatory Platelet Functions.

OBJECTIVE: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets' inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s-1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. CONCLUSIONS: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Effect of Endothelial Culture Medium Composition on Platelet Responses to Polymeric Biomaterials.

Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.

In vitro and in vivo blood compatibility of concentrated polymer brushes.

Concentrated polymer brushes (CPBs) and semi-dilute polymer brushes (SDPBs) of poly(2-hydroxyethyl methacrylate), poly(2-hydroxyethyl acrylate), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA) and poly(2-methoxyetyl acrylate) were prepared on silica particles and silicon wafers by surface-initiated atom transfer radical polymerization (SI-ATRP). In order to evaluate in vitro blood compatibility, plasma protein adsorption on the brushes was quantified with a BCA protein assay, and the adsorbed proteins on the brushes were identified using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). All four CPBs displayed much less protein adsorption than their corresponding SDPBs. Interestingly, the number and type of identified proteins differed on the brushes. Platelet adhesion was then examined on the brushes, whereby CPBs suppressed platelet adhesion to a greater extent than the corresponding SDPBs, although platelet activation was observed on all surfaces. As a result, the CPBs of PPEGMA prevented platelet adhesion the most. After screening the polymers by in vitro evaluation, CPBs of PPEGMA were then grafted on a catheter by SI-ATRP. The catheter with the CPBs was implanted into the jugular vein of a rabbit. The in vivo assessment after three weeks of implantation confirmed that the CPBs caused little coagulation or inflammation, whereas the pristine catheter exhibited inflammation and encapsulation.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

The strategy of modulation blood responses by surface modification with different functional groups on polyester film.

A main problem in the design of blood-contacting biomaterials has been the deficiency of a systematic understanding of blood-biomaterial interactions and the strategy to modulate blood responses. In this work, different functional groups including carboxyl (COOH), hydroxyl (OH) and zwitterionic sulfobetaine group (⊕N((CH3 )2 )(CH2 )3 SO3-○- , SMDB) were grafted on the poly (butylene terephthalate) (PBT) film to study how the functional groups modulate blood responses and in terms of interaction with the coagulation system, the complement system, and platelets. The results showed protein absorption and platelet adhesion was stronger on the PBT bearing COOH group than PBT films bearing OH and zwitterionic sulfobetaine groups (total protein (µg/cm2 ): 32.92 ± 5.89 vs. 22.02 ± 1.44 vs. 19.09 ± 1.59; platelet adhesion (/mm2 ): 1,626.7 ± 120.1 vs. 1,395.6 ± 363.3 vs. 1,102.2 ± 373.7), which had a rougher and negatively charged surface, and the coagulation system was inhibited by binding fibrinogen (Fg) and coagulation factors. Meanwhile, PBT-PSMDB showed anticoagulant property and induced platelet activation. As a result, complement formation on these two films were less than PBT bearing OH groups by inhibiting the coagulation system (C3a (ng/ml): 3,745.4 ± 143.9 vs. 3,290.9 ± 249.7 vs. 4,887.9 ± 88.9; C5a (ng/ml): 22.1 ± 2.6 vs. 22.3 ± 1.8 vs. 27.9 ± 2.0). On the other hand, PBT bearing OH groups did not facilitate remarkable platelet adhesion and activation, and had no influence on platelet aggregation, hypotonic shock response, and coagulation system. The above results showed that the blood responses were highly interlinked, and could be modulated by grafting with different functional groups on the biomaterial surfaces. These findings may help identify a strategy to design materials with better hemocompatibility for blood contact, filtration, and purification applications.

Investigating the hemostatic effect of medicinal plant Arnebia euchroma (Royle) I.M.Johnst extract in a mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Arnebia euchroma (Royle) I.M.Johnst (AE) has been reported to be a potentially useful medicinal herb for the treatment of several circulatory diseases in traditional Chinese medicine. It shows effects such as "cooling of the blood," promotion of blood circulation, detoxification, and rash clearance. AIM OF THE STUDY: To explore the hemostatic effect of the ethyl acetate extract of AE in mice. MATERIALS AND METHODS: In this study, we explored the effects of AE on bleeding time, blood coagulation time, platelet count, and blood coagulation parameters in normal Kunming mice. Different doses of the AE extract (5, 10, and 20 g kg-1·day-1) were administered to mice for 14 days. Sodium carboxymethyl cellulose (CMC-Na at 0.5%) and Yunnan Baiyao (0.8 g kg-1·day-1) were administered as negative and positive control treatments, respectively. Bleeding time, blood coagulation time, platelet count, blood platelet aggregation, blood platelet adhesion to fibrinogen, platelet factor 4 (PF-4) secretions from blood platelets, and blood coagulation parameters including prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen (FIB) levels were measured on day 15 of administration. RESULTS: Bleeding and blood coagulation time were significantly lower and TT was shorter in the AE extract-treated groups than in the control groups. Furthermore, FIB levels and platelet count were higher, whereas blood platelet aggregation, blood platelet adhesion to fibrinogen, and PF-4 secretion from blood platelets were more obvious in the AE extract-treated groups than in the control group. However, no significant differences were detected for PT and aPTT between the extract-treated and control groups. CONCLUSIONS: The ethyl acetate extract of AE showed potential hemostasis effects in mice by shortening the bleeding and coagulation time. In addition, the extract increased platelet count and induced blood platelet aggregation, blood platelet adhesion to fibrinogen, PF-4 secretion from blood platelets, and FIB level, while it shortened TT.

Targeting Gα13-integrin interaction ameliorates systemic inflammation.

Systemic inflammation as manifested in sepsis is an excessive, life-threatening inflammatory response to severe bacterial or viral infection or extensive injury. It is also a thrombo-inflammatory condition associated with vascular leakage/hemorrhage and thrombosis that is not effectively treated by current anti-inflammatory or anti-thrombotic drugs. Here, we show that MB2mP6 peptide nanoparticles, targeting the Gα13-mediated integrin "outside-in" signaling in leukocytes and platelets, inhibited both inflammation and thrombosis without causing hemorrhage/vascular leakage. MB2mP6 improved mouse survival when infused immediately or hours after onset of severe sepsis. Furthermore, platelet Gα13 knockout inhibited septic thrombosis whereas leukocyte Gα13 knockout diminished septic inflammation, each moderately improving survival. Dual platelet/leukocyte Gα13 knockout inhibited septic thrombosis and inflammation, further improving survival similar to MB2mP6. These results demonstrate that inflammation and thrombosis independently contribute to poor outcomes and exacerbate each other in systemic inflammation, and reveal a concept of dual anti-inflammatory/anti-thrombotic therapy without exacerbating vascular leakage.