Cyclic thrombocytopenia associated with estradiol: a case report.

OBJECTIVES: Cyclic thrombocytopenia (CTP) is a rare blood disorder characterized by periodic fluctuations in platelet counts. CTP usually appears in pre-menopausal women, and these fluctuations of platelets are in phase with the menstrual cycle. CTP is a heterogeneous disease, and the pathogenic mechanism is still unclear. Therefore, it harbors great significance for exploring the association of fluctuations in platelet counts with hormonal-cycle. MATERIALS: Firstly, we washed human platelets from healthy volunteers following the Declaration of Helsinki. Flow cytometer was employed to measure the mitochondrial inner transmembrane potential (ΔΨm) depolarization, PS exposure, P-selectin expression, and GPIIb/IIIa activation in platelets. In addition, western blot detected the related protein expression. The corresponding assay kit measured the caspase-3 and PDE3A activity. Finally, flow cytometry determined mouse platelets labeled with calcein. RESULTS: We find a reverse relationship between the platelet count and serum estradiol (E2) level in a CTP patient. We demonstrated that E2 induces platelet apoptosis in vitro and platelet clearance in vivo. We further discovered that E2 activates phosphodiesterase 3A, which inhibits protein kinase A (PKA), leading to PKA-mediated platelet apoptosis. Activation of PKA protected platelets from E2-induced thrombocytopenia and elevated the number of mice circulatory platelets. CONCLUSIONS: We find that E2 induces platelet apoptosis and clearance through PDE3A-mediated PKA inhibition. Activation of PKA rescues E2-induced thrombocytopenia in mice. Thus, our study reveals a pathogenesis of E2-related CTP and suggests promising therapeutic strategies for the disease.

Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression.

Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.

Antagonizing αvß3 Integrin Improves Ischemia-Mediated Vascular Normalization and Blood Perfusion by Altering Macrophages.

Background: αVß3 integrin has been implicated in the physiological processes and pathophysiology of important angiogenesis-related disorders; however, the preclinical and clinical data on integrin αVß3 antagonists have not demonstrated improved outcomes. Our goal was to test the hypothesis that inhibition of αVß3 integrin improves blood flow in a mouse hindlimb ischemia model. Methods: In this study, we examined the effect of cilengitide, an αVß3/αVß5 integrin-specific RGD-mimetic cyclic peptide, on blood perfusion and angiogenesis after hindlimb ischemia. Blood flow was measured using Laser Doppler Scanner. Vascular density, and macrophages infiltration were examined by immunofluorescence. Macrophage polarization was measured by quantitative real time PCR. Results: We found that low-dose, not high-dose, cilengitide increased blood flow perfusion, capillary formation, and pericyte coverage, accompanied by an accumulation of macrophages and increased expression of the chemokine (C-C motif) ligand 2 (CCL2) in ischemic muscles. Macrophage depletion using clodronate liposomes resulted in a reduction in low-dose cilengitide-induced blood flow perfusion, macrophage accumulation, pericyte coverage, and CCL2 expression. Finally, in vitro assays showed that low-dose, not high-dose, cilengitide increased macrophage migration. Conclusion: These studies identified a novel role of the inhibition of αVß3 integrin in modulating ischemia-induced angiogenesis, possibly through effects on macrophage infiltration and polarization, and revealed αVß3 integrin inhibition to be a promising therapeutic strategy for peripheral artery disease.

Engineered Nanoplatelets for Targeted Delivery of Plasminogen Activators to Reverse Thrombus in Multiple Mouse Thrombosis Models.

Rapid cut-off of blood supply in diseases involving thrombosis is a major cause of morbidity and mortality worldwide. However, the current thrombolysis strategies offer limited results due to the therapeutics' short half-lives, low targeting ability, and unexpected bleeding complications. Inspired by the innate roles of platelets in hemostasis and pathological thrombus, platelet membrane-camouflaged polymeric nanoparticles (nanoplatelets) are developed for targeting delivery of the thrombolytic drug, recombinant tissue plasminogen activator (rt-PA), to local thrombus sites. The tailor-designed nanoplatelets efficiently accumulate at the thrombi in pulmonary embolism and mesenteric arterial thrombosis model mice, eliciting a significantly enhanced thrombolysis activity compared to free rt-PA. In addition, the nanoplatelets exhibit improved therapeutic efficacy over free rt-PA in an ischemic stroke model. Analysis of in vivo coagulation indicators suggests the nanoplatelets might possess a low risk of bleeding complications. The hybrid biomimetic nanoplatelets described offer a promising solution to improve the efficacy and reduce the bleeding risk of thrombolytic therapy in a broad spectrum of thrombosis diseases.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Corrigendum: Transplantation of Normal Adipose Tissue Improves Blood Flow and Reduces Inflammation in High Fat Fed Mice With Hindlimb Ischemia.

[This corrects the article on p. 197 in vol. 9, PMID: 29568274.].

Transplantation of Normal Adipose Tissue Improves Blood Flow and Reduces Inflammation in High Fat Fed Mice With Hindlimb Ischemia.

Background: Fat deposition is associated with peripheral arterial disease. Adipose tissue has recently been implicated in vascular remodeling and angiogenic activity. We hypothesized that the transplantation of adipose tissues from normal mice improves blood flow perfusion and neovascularization in high-fat diet fed mice. Methods: After 14 weeks of high-fat diet (HFD)-fed mice, unilateral hind limb ischemia was performed. Subcutaneous white adipose tissue (WAT) and brown adipose tissue (BAT) fat pads were harvested from normal EGFP mice, and subcutaneously transplanted over the region of the adductor muscles of HFD mice. Blood flow was measured using Laser Doppler Scanner. Vascular density, macrophages infiltration, and macrophage polarization were examined by RT-qPCR, and immunohistochemistry. Results: We found that the transplantation of WAT derived from normal mice improved functional blood flow in HFD-fed mice compared to mice transplanted with BAT and sham-treated mice. WAT transplantation increased the recruitment of pericytes associated with nascent blood vessels, but did not affect capillary formation. Furthermore, transplantation of WAT ameliorated HFD-induced insulin resistance, M2 macrophage predominance and the release of arteriogenic factors in ischemic muscles. Mice receiving WAT also displayed a marked reduction in several proinflammatory cytokines. In contrast, mice transplanted with BAT were glucose intolerant and demonstrated increased IL-6 levels in ischemic muscles. Conclusion: These results indicate that transplantation of adipose tissue elicits improvements in blood perfusion and beneficial effects on systemic glucose homeostasis and could be a promising therapeutic option for the treatment of diabetic peripheral arterial disease.

Sodium tanshinone IIA sulfonate prevents lipopolysaccharide-induced inflammation via suppressing nuclear factor-κB signaling pathway in human umbilical vein endothelial cells.

Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA, has been demonstrated to have potent anti-inflammatory properties. However, the protective effects of STS on lipopolysaccharide (LPS)-induced inflammation in endothelial cells remain to be elucidated. In the present study, human umbilical vein endothelial cells (HUVECs) were used to explore the effects of STS on LPS-induced inflammation and the molecular mechanism involved. HUVECs were pretreated with STS for 2 h, followed by stimulation with LPS. Then expression and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, and the activation of nuclear factor-κB (NF-κB) were assessed. The results demonstrated that STS significantly decreased LPS-induced TNF-α and IL-1ß protein expression in HUVECs. Similarly, the increased levels of TNF-α and IL-1ß in cell supernatants stimulated by LPS were also significantly inhibited by STS. Furthermore, STS inhibited LPS-induced NF-κB p65 phosphorylation and nuclear translocation. All the results suggest that STS prevents LPS-induced inflammation through suppressing NF-κB signaling pathway in endothelial cells, indicating the potential utility of STS for the treatment of inflammatory diseases.

Polydatin Prevents Methylglyoxal-Induced Apoptosis through Reducing Oxidative Stress and Improving Mitochondrial Function in Human Umbilical Vein Endothelial Cells.

Methylglyoxal (MGO), an active metabolite of glucose, has been reported to induce vascular cell apoptosis in diabetic complication. Polydatin (PD), a small natural compound from Polygonum cuspidatum, has a number of biological functions, such as antioxidative, anti-inflammatory, and nephroprotective properties. However, the protective effects of PD on MGO-induced apoptosis in endothelial cells remain to be elucidated. In this study, human umbilical vein endothelial cells (HUVECs) were used to explore the effects of PD on MGO-induced cell apoptosis and the possible mechanism involved. HUVECs were pretreated with PD for 2 h, followed by stimulation with MGO. Then cell apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) impairment, mitochondrial morphology alterations, and Akt phosphorylation were assessed. The results demonstrated that PD significantly prevented MGO-induced HUVEC apoptosis. PD pretreatment also significantly inhibited MGO-induced ROS production, MMP impairment, mitochondrial morphology changes, and Akt dephosphorylation. These results and the experiments involving N-acetyl cysteine (antioxidant), Cyclosporin A (mitochondrial protector), and LY294002 (Akt inhibitor) suggest that PD prevents MGO-induced HUVEC apoptosis, at least in part, through inhibiting oxidative stress, maintaining mitochondrial function, and activating Akt pathway. All of these data indicate the potential application of PD for the treatment of diabetic vascular complication.

Platelet-Derived Factor V Is a Critical Mediator of Arterial Thrombosis.

BACKGROUND: Coagulation factor V (FV) plays a key role in hemostasis, is present in plasma and platelets, and has both pro- and anticoagulant properties; however, the contribution of platelet-derived FV to arterial thrombosis remains undetermined. METHODS AND RESULTS: Using transgenic mice with various levels of FV gene expression that was restricted to the plasma or platelets, the roles of platelet FV were evaluated in the regulation of arterial thrombosis and platelet activation. Mice with higher levels of platelet FV exhibited faster thrombotic occlusion of the carotid artery after injury compared with mice with lower platelet FV levels. Infusion of platelets with higher levels of FV into transgenic mice with undetectable levels of platelet FV reduced the time to carotid artery occlusion. In contrast, infusion of purified recombinant plasma FV into mice with undetectable platelet FV levels failed to reduce the carotid occlusion times following injury. Evaluation of isolated platelets revealed that platelet-derived FV was critical for the regulation of platelet activation. These effects were associated with an increased level of expression of P-selectin and increased cGMP in platelets. CONCLUSIONS: We established that platelet-derived FV is a critical mediator of arterial thrombosis that involves platelet activation.

Hydroxysafflor Yellow A Promotes Angiogenesis via the Angiopoietin 1/ Tie-2 Signaling Pathway.

BACKGROUND: The flowers of Carthamus tinctorius L. are widely used in traditional Chinese medicine to treat cerebrovascular and cardiovascular diseases. Hydroxysafflor yellow A (HSYA), the main constituent of C. tinctorius L. flowers, is known for its multiple biological activities. The present study investigated the effects of HSYA on angiogenesis in vitro and in a mouse hindlimb ischemia model. METHODS: Using human umbilical vein endothelial cells (HUVEC) in vitro and a mouse hindlimb ischemia model in vivo, the angiogenic role of HSYA was evaluated. RESULTS: HSYA significantly increased the capillary-like tube formation and migration of HUVEC. HSYA not only induced a rise in the expression of angiopoietin 1 and Tie-2 but it also increased phosphorylation of Tie-2, Akt, and extracellular signal-regulated kinase 1/2. Furthermore, an anti-Tie-2 neutralizing antibody significantly inhibited HSYA-induced HUVEC tube formation and migration. In vivo, the recovery of perfusion of ischemic hindlimb tissue after femoral artery interruption was significantly increased in HSYA-treated mice compared to vehicle controls. Consistent with these results, the arteriole and capillary densities in ischemic gastrocnemius muscles were significantly increased in HSYA-treated mice. CONCLUSIONS: These results indicate the potential utility of HSYA for the treatment of ischemic diseases.

[Methylglyoxal inhibits human umbilical vein cell migration in vitro by down-regulating integrinß3].

OBJECTIVE: To explore the effects of methylglyoxal on endothelia cell migration. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated by serial concentrations of methylglyoxal (MGO, 0, 25, 50, 100 and 200 µmol/L) for 24 h, and the cell migration was assessed by scratch wound and Transwell assay. The expression of integrin ß3 in the treated cells was examined by immunoblotting, and the effect of an anti-ß3 antibody, LM609, on cell migration was investigated. RESULTS: Methylglyoxal significantly inhibited HUVEC migration in a concentration-dependent manner (P<0.05). Methylglyoxal decreased the expression of integrin ß3 in a time- and concentration-dependent manner (P<0.05). LM609 also significantly inhibited HUVEC migration (P<0.05). CONCLUSION: Methylglyoxal inhibits HUVEC migration in vitro by down-regulating integrin ß3 expression.