The profibrotic cytokine transforming growth factor‐β1 increases endothelial progenitor cell angiogenic properties

Summary. Background: Transforming growth factor‐β1 (TGF‐β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF‐β1 modulation of EPC angiogenic properties. Methods: TGF‐β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF‐β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony‐forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF‐β1 in ECFCs by using short interfering RNA. Results: Total TGF‐β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF‐β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF‐β1 receptors. Conclusions: TGF‐β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF‐β1 may play a role during vascular remodeling in fibrotic disease states via EPCs.     

Phosphoinositide 3‐kinase mediates CD40 ligand‐induced oxidative stress and endothelial dysfunction via Rac1 and NADPH oxidase 2

Summary. Objectives: CD40 ligand (CD40L) has been implicated as an inducer of reactive oxygen species (ROS) generation in endothelial cells, but definitive evidence for this and the in vivo relevance haves not been demonstrated fully. We thus investigated whether phosphoinositide 3‐kinase (PI3K) was linked to ROS generation and endothelial reactivity in response to CD40L. Methods and Results: CD40L treatment activated PI3K activity by regulating the association between PI3K p85 and the CD40 receptor. CD40L exposure also stimulated the GTPase Rac1, which is known to activate NADPH oxidases, and enhanced ROS formation, whereas PI3K inhibition or depletion by small interfering RNA (siRNA) prevented these responses. Subsequently, PI3K overexpression activated Rac1 and increased ROS generation. These responses were not observed in the presence of inactive Rac1 or siRNA against the NADPH oxidase subunit NOX2. Protein kinase Cζ mediates PI3K‐regulated NADPH oxidase activation by promoting cellular p47phox translocation. Importantly, PI3K inhibition prevented CD40L‐mediated ROS generation and endothelial dysfunction in a mouse model. In summary, PI3K mediates CD40L‐induced ROS production and subsequent endothelial dysfunction. Conclusions: Targeting PI3K may provide a new therapeutic approach in diseases associated with oxidative stress and endothelial dysfunction.     

Co‐culture of adipose‐derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co‐implantation of adipose‐derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co‐cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell–fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co‐implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary‐like structures by co‐implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary‐like structures near the top of the construct, indicating that perfusion of the cell–fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3‐kinase‐β signaling

Summary. Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G‐protein‐coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real‐time‐PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y₁ receptor. Light transmission aggregation experiments showed dose‐dependent aggregation induced by succinate, reaching a maximum response at 0.5 mm . The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb–IIIa and P‐selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3‐kinase‐β activation, and receptor desensitization. Furthermore, succinate‐induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A₂, and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y₁₂ receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate‐induced platelet aggregation depends on thromboxane A₂ generation, ATP release, and P2Y₁₂ activation.     

Granulocyte colony‐stimulating factor (G‐CSF) depresses angiogenesis in vivo and in vitro: implications for sourcing cells for vascular regeneration therapy

Summary. Background: The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G‐CSF administration. Objective: To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G‐CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Patients/Methods: Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G‐CSF‐mobilized samples were collected before administration, at apheresis harvest, and at follow‐up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony‐forming unit (CFU)‐EPC and EOC assays. The effect of exogenous G‐CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Results: Our data show that G‐CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU‐EPCs. Furthermore, G‐CSF administration did not mobilize CD34+CD133? cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G‐CSF inhibited CFU‐EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. Conclusions: G‐CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G‐CSF may come only from a paracrine effect from HPCs.     

Characteristics of recombinant W501S mutated human γ‐glutamyl carboxylase

Summary. A mutation (W501S) in the vitamin K‐dependent γ‐glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC‐W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495–513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d‐OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high‐affinity site for d‐OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the K_M for vitamin K hydroquinone in wild‐type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.     

The glycoprotein Ibα–von Willebrand factor interaction induces platelet apoptosis

Summary. Background: The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbα–VWF interaction. Objectives: To investigate whether the GPIbα–VWF interaction induces platelet apoptosis and the role of 14‐3‐3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild‐type (1b9) or mutant GPIb–IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin‐induced GPIbα–VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear‐induced apoptosis was eliminated by the anti‐GPIbα antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb–IX with GPIbα truncated at residue 551 or a serine‐to‐alanine mutation at the 14‐3‐3ζ‐binding site in GPIbα. Conclusions: This study demonstrates that the GPIbα–VWF interaction induces apoptotic events in platelets, and that the association of 14‐3‐3ζ with the cytoplasmic domain of GPIbα is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.     

Thrombin upregulates the angiopoietin–Tie2 Axis: endothelial protein C receptor occupancy prevents the thrombin mobilization of angiopoietin 2 and P‐selectin from Weibel–Palade bodies

Summary. Background: Activated protein C (APC) in complex with endothelial protein C receptor (EPCR) can reverse the barrier‐disruptive and cytotoxic effects of proinflammatory cytokines by cleaving protease‐activated receptor 1 (PAR‐1). Recently, it was reported that the PAR‐1‐dependent vascular barrier‐protective effect of APC is mediated through transactivation of the angiopoietin (Ang)–Tie2 signaling pathway. The antagonist of this pathway, Ang2, is stored in Weibel–Palade bodies within endothelial cells. Objectives: To determine whether the occupancy of EPCR by its ligand can switch the PAR‐1‐dependent signaling specificity of thrombin through the Ang–Tie2 axis. Methods: We activated endothelial cells with thrombin before and after treating them with the catalytically inactive Ser195→Ala substitution mutant of protein C. The expression levels of Ang1, Ang2 and Tie2 in response to thrombin were measured by both an enzyme‐linked immunosorbent assay and a cell permeability assay in the absence and presence of small interfering RNA and a blocking antibody to Tie2. Results: Thrombin upregulated the expression of both Ang1 and Tie2 but downregulated the expression of Ang2 when EPCR was occupied by its ligand. The Ang1–Tie2‐dependent protective effect of thrombin was initiated through protein C inhibiting the rapid mobilization of Ang2 from Weibel–Palade bodies. Interestingly, the protein C mutant also inhibited the thrombin mobilization of P‐selectin. Conclusions: These results suggest a physiologic role for the low concentration of thrombin in maintaining the integrity of the EPCR‐containing vasculature through the PAR‐1‐dependent inhibition of Ang2 and P‐selectin release from Weibel–Palade bodies.     

Flow mediated vasodilation and circulating concentrations of high sensitive C‐reactive protein,interleukin‐6 and tumor necrosis factor‐α in normal pregnancy – The Cardiovascular Risk in Young Finns Study

Background: Traditional risk factors such as hyperlipidemia induce a state of inflammation that impairs vascular function. Despite marked maternal hyperlipidemia, endothelial function improves during pregnancy. In non‐pregnant state increased circulating levels of pro‐inflammatory cytokines and high sensitive C‐reactive protein (hsCRP) lead to attenuated flow mediated vasodilation. Relation between endothelial function and pro‐inflammatory cytokines has not been studied thoroughly in pregnancy. The aim of this study was to evaluate the effect of pregnancy on hsCRP and pro‐inflammatory cytokines and their associations with vascular endothelial function. Methods: As part of population‐based, prospective cohort Cardiovascular Risk in Young Finns study conducted in Finland we measured brachial artery flow mediated dilation (FMD) and serum concentrations of hsCRP, interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) in 57 pregnant Finnish women throughout gestation and 62 control women matched for age and smoking. Results: HsCRP‐concentration was greater in pregnancy compared to non‐pregnant controls (median hsCRP 2·52 mg l^?1 versus 1·21 mg l^?1, P<0·001). IL‐6‐concentration was slightly increased in pregnancy compared with the non‐pregnant controls (median 1·66 versus 1·32 mg l^?1, non‐significant [NS]) and TNF‐α‐concentration was slightly decreased in pregnant group (2·11 versus 2·38 pg ml^?1, NS). FMD increased during pregnancy and IL‐6 had a positive correlation to the FMD in pregnancy (R = 0·288, P = 0·031). Conclusions: Improvement of FMD in normal pregnancy was not affected by increase in hsCRP concentration. We found an association with IL‐6 and FMD but we believe that improvement in endothelial function during normal pregnancy is not caused by variation in hsCRP, IL‐6 or TNF‐α.     

The α‐granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome

See also García Á. Clinical proteomics in platelet research: challenges ahead. This issue, pp 1784–5. Summary. Background: Deficiencies in granule‐bound substances in platelets cause congenital bleeding disorders known as storage pool deficiencies. For disorders such as gray platelet syndrome (GPS), in which thrombocytopenia, enlarged platelets and a paucity of α‐granules are observed, only the clinical and histologic states have been defined. Objectives: In order to understand the molecular defect in GPS, the α‐granule fraction protein composition from a normal individual was compared with that of a GPS patient by mass spectrometry (MS). Methods: Platelet organelles were separated by sucrose gradient ultracentrifugation. Proteins from sedimented fractions were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis, reduced, alkylated, and digested with trypsin. Peptides were analyzed by liquid chromatography–tandem MS. Mascot was used for peptide/protein identification and to determine peptide false‐positive rates. Mass Sieve was used to generate and compare parsimonious lists of proteins. Results: As compared with control, the normalized peptide hits (NPHs) from soluble, biosynthetic α‐granule proteins were markedly decreased or undetected in GPS platelets, whereas the NPHs from soluble, endocytosed α‐granule proteins were only moderately affected. The NPHs from membrane‐bound α‐granule proteins were similar in normal platelets and GPS platelets, although P‐selectin and Glut3 were slightly decreased, consistent with immunoelectron microscopy findings in resting platelets. We also identified proteins not previously known to be decreased in GPS, including latent transforming growth factor‐β‐binding protein 1(LTBP1), a component of the transforming growth factor‐β (TGF‐β) complex. Conclusions: Our results support the existence of ‘ghost granules’ in GPS, point to the basic defect in GPS as failure to incorporate endogenously synthesized megakaryocytic proteins into α‐granules, and identify specific new proteins as α‐granule inhabitants.     

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti‐β₂‐glycoprotein I (β₂‐GPI) autoantibodies. β₂‐GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish‐hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β₂‐GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes‐derived proteins to induce anti‐β₂‐GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen‐like protein A [SclA], and streptococcal collagen‐like protein B [SclB]) were found to interact with β₂‐GPI. Only binding to protein H induces a conformational change in β₂‐GPI, thereby exposing a cryptic epitope for APS‐related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody‐related epitope in domain I of β₂‐GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti‐protein H antibodies also generated anti‐β₂‐GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in β₂‐GPI, resulting in the formation of antiβ₂‐GPI autoantibodies. This constitutes a novel mechanism for the formation of anti‐β₂‐GPI autoantibodies.     

Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2‐antiplasmin

Summary. Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of α₂‐antiplasmin (α₂AP) incorporated within fibrin. Circulating prolyl‐specific serine proteinase, antiplasmin‐cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met‐α₂AP between ‐Pro12‐Asn13‐ to yield Asn‐α₂AP, which is crosslinked to fibrin approximately 13× more rapidly than Met‐α₂AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met‐α₂AP to Asn‐α₂AP and thereby enhance endogenous fibrinolysis. Methods and results: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl‐Arg‐(8‐amino‐3,6‐dioxaoctanoic acid)‐d ‐Ala‐l ‐boroPro selectively inhibited APCE vs. DPPIV, with an apparent K_i of 5.7 nm vs. 6.1 μm , indicating that an approximately 1000‐fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K_i of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose‐dependent decrease of APCE‐mediated Met‐α₂AP cleavage, which ultimately shortened plasminogen activator‐induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC₅₀ value in plasma remaining comparable to that in phosphate buffer. Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3‐kinase

Summary. Background and objective: Salvianolic acid A (SAA) is a water‐soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. Methods and Results: SAA dose‐dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP‐enhanced platelet P‐selectin expression and fibrinogen binding, which consequently hampered ADP‐induced platelet–leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside‐in signaling. Under an arterial shear rate of 1000 s^?1, SAA decreased platelet adhesion on collagen surfaces by ～ 40%. Western blot analysis showed that SAA, like the phosphoinositide 3‐kinase (PI3K) inhibitors LY294002 and TGX‐221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild‐type mice (35 ± 2 min without SAA and 56 ± 4 min with SAA; P < 0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in Ldlr^tm1Her mutant mice (21 ± 2 min without SAA and 45 ± 4 min with SAA; P < 0.01). Conclusions: SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.