Essential role of glycoprotein Ibα in platelet activation.

Glycoprotein (GP) Ib, the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIb interacts with 14-3-3, regulate ng the VWF-GPIb-elicited signal transduction and VWF binding function of GPIb. However, we unexpectedly found that the GPIb-14-3-3 association, beyond VWF-dependent function, is essential for general platelet activation. We found that the GPIb cytoplasmic tail peptide MPC, a potential GPIb inhibitor, by itself induced platelet aggregation, integrin αIIbß3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIb in mouse platelets (10aa-/-) decreased platelet aggregation, integrin IIb3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPC-treated platelets. MPC-induced platelet activation was abolished by the pan-PKC inhibitor and PKC deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa-/- platelets. GPIb regulates PKC activity by sequestering 14-3-3 from PKC. In vivo, the deletion of the GPIb cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIb cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF-GPIb axis.

Mutation detection and inhibitor analysis of 43 children with severe hemophilia A in a single center: three novel mutations.

To investigate the risk factors of FVIII inhibitors development in severe hemophilia A (HA) patients who were received on-demand therapy and were infused with plasma cryoprecipitate and multiple FVIII concentrates alternately. We collected clinical information from 43 severe HA children who were treated with plasma cryoprecipitate and multiple FVIII concentrates. The F8 mutation was detected by long-distance PCR for inversion and detected by all exons and their flanking sequencing for other mutations. The inhibitor detection was performed by Nijmegen-modified Bethesda assay. The impact of novel amino substitutions on FVIII protein was predicted by SIFT and PolyPhen-2. The 3D analysis of missense mutations was performed using Swiss-PdbViewer. FVIII inhibitors were detected in nine cases (20.9%). All of the inhibitor positive cases had high risk F8 gene mutations. In most of the positive cases (7/9), inhibitors were developed during the first 10 EDs, which was significantly higher than that in the 10-50 EDs group and 50 EDs group (p = 0.009). Three novel mutations were reported, including c.214G > T (E72X), c.218 T > C (F73S), and c.2690C > G (S840X). For severe HA patients who are treated with multiple products of replacement therapy, it is important to supervise inhibitor during the first 10EDs, especially for those with high risk F8 gene mutations. F8 gene mutation is one of the most important genetic factors for inhibitor development. It is essential to detect F8 gene for all severe HA patients. Three novel mutations were reported to expand the mutation spectrum of the F8 gene.

Novel GNE missense variants impair de novo sialylation and cause defective angiogenesis in the developing brain in mice.

ABSTRACT: Glucosamine (UDP-N-acetyl)-2-epimerase and N-acetylmannosamine (ManNAc) kinase (GNE) is a cytosolic enzyme in de novo sialic acid biosynthesis. Congenital deficiency of GNE causes an autosomal recessive genetic disorder associated with hereditary inclusion body myopathy and macrothrombocytopenia. Here, we report a pediatric patient with severe macrothrombocytopenia carrying 2 novel GNE missense variants, c.1781G>A (p.Cys594Tyr, hereafter, C594Y) and c.2204C>G (p.Pro735Arg, hereafter, P735R). To investigate the biological significance of these variants in vivo, we generated a mouse model carrying the P735R mutation. Mice with homozygous P735R mutations exhibited cerebral hemorrhages as early as embryonic day 11 (E11), which subsequently progressed to large hemorrhages in the brain and spinal cord, and died between E11.5 and E12.5. Defective angiogenesis such as distended vascular sprouts were found in neural tissues and embryonic megakaryocytes were abnormally accumulated in the perineural vascular plexus in mutant mouse embryos. Furthermore, our in vitro experiments indicated that both C594Y and P735R are loss-of-function mutations with respect to de novo sialic acid biosynthesis. Overall, this study reveals a novel role for GNE-mediated de novo sialic acid biosynthesis in mouse embryonic angiogenesis.

A conserved ZFX/WNT3 axis modulates the growth and imatinib response of chronic myeloid leukemia stem/progenitor cells.

BACKGROUND: Zinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear. METHODS: Real-time quantitative PCR (RT-qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT-qPCR and western blotting were used to study the effect of ZFX on ß-catenin signaling. RESULTS: We showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/ß-catenin signaling, including c-MYC and CCND1 expression. CONCLUSION: The present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells.

[Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide].

OBJECTIVE: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. METHODS: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. RESULTS: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. CONCLUSION: Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.

[Application of Bionano-DNA Full-Length Optical Mapping Technology in Genetic Diagnosis of Hemophilia A Carriers].

OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.

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.

Safety and efficacy of an anti-human APC antibody for prophylaxis of congenital factor deficiencies in preclinical models.

Rebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.

[Recent Advances in SARS-CoV-2-Induced Immune Thrombocytopenia --Review].

SARS-CoV-2-induced immune thrombocytopenia (SARS-CoV-2-induced ITP) is an autoimmune disease secondary to virus infections. Its diagnosis is often based on exclusion of other possible causes of thrombocytopenia in COVID-19 patients. Common laboratory examinations include coagulation function, thrombopoietin and drug-dependent antibodies. Since both bleeding and thrombosis risks are seen in SARS-CoV-2-induced ITP patients, individual remedy is essential for the treatment of this disease. Because thrombopoietin receptor agonist(TPO-RA) has the side effect of accelerating thrombosis and may aggravate the pulmonary embolism symptoms of patients, it should be used for refractory SARS-CoV-2-induced ITP patients only. This review briefly summarizes the recent research progress in the pathogenesis, diagnosis and treatment of SARS-CoV-2-induced ITP.

Appropriate pre-transplant strategy for patients with myelodysplastic syndromes receiving allogeneic haematopoietic stem cell transplantation after myeloablative conditioning.

Purpose: Appropriate pre-transplant strategies in patients with myelodysplastic syndromes (MDS) remain challenging. We sought to assess the effect of different pre-transplant therapies and transplantation interval times on patient prognosis. Methods: We retrospectively analysed clinical data for 371 consecutive MDS patients after myeloablative transplantation between 2007 and 2019. Results: The median age of the patients was 38 years (range, 12-64 years). A total of 114 patients (31%) received supportive care (SC), 108 (29%) hypomethylating agents (HMAs), and 149 (40%) chemotherapy-based therapy before transplantation. In patients who received HMA or SC, there was no significant difference in overall survival (OS; P=0.151) or relapse-free survival (RFS; P=0.330), except that HMA-treated patients had a lower rate of non-relapse mortality (5-year NRM: 18% vs. 32%, P=0.035). However, compared with patients who received HMA, those who received chemotherapy-based therapy had a lower 5-year OS rate (56% vs. 69%, P=0.020) and a slightly higher 5-year NRM rate (28% vs. 18%, P=0.067). Compared to the delayed transplant group (transplant interval ≥6 months), the early transplant group (transplant interval <6 months) had a superior 5-year OS (66% vs. 51%, P=0.001) and a lower 5-year cumulative incidence of NRM (22% vs. 36%, P=0.001). Conclusion: The findings of the study indicate that receiving an appropriate pre-transplant strategy (SC/HMA + <6 months) significantly improves OS and decreases NRM in MDS patients after myeloablative transplantation.

Insights from a rare myeloproliferative neoplasm with coexisting BCR-ABL1 fusion gene, CALR, and TET2 mutations treated with nilotinib and ruxolitinib.

Myeloproliferative neoplasms (MPNs) with concurrent BCR-ABL1 fusion gene and CALR mutation are especially rare. We report a patient with coexisting BCR-ABL1 fusion gene, CALR, and TET2 mutations who was treated with the combination of the second-generation TKI nilotinib and JAK1/JAK2 inhibitor ruxolitinib.

[The Effect of VWF Propeptide on VWF Mutant in D1 Domain].

OBJECTIVE: To investigate whether co-transfection of wild-type VWFpp with VWF mutant in D1 region is able to correct VWF defects in biosynthesis and secretion. METHODS: Four VWF mutant plasmids were single transfected into HEK 293 cells, or co-transfected into HEK 293 cells with the wild type VWFpp plasmids. The VWF in supernatant and lysate of transfected cells were analyzed by ELISA, vertical VWF multimer electrophoresis. The retention of VWF in endoplasmic reticulum of transfected cells were detected by immunofluorescence confocal microscope. RESULTS: In the vertical VWF multimer analysis, with co-expressing VWF mutant and VWFpp, the VWF multimer bands disappeared, and the VWF antigen in both supernatant and lysate of cells decreased, compared with the single expression of VWF mutant. Although the intracellular levels of VWF antigens decreased after co-expression, the retention rate of VWF mutant decreased in endoplasmic reticulum. CONCLUSION: VWFpp can reduce the retention of VWF in endoplasmic reticulum, assists the transport of VWF between subcellular organelles. However, VWFpp inhibits the biosynthesis and secretion of VWF about the mutant in D1 domain.

Bifunctional effect of the inflammatory cytokine tumor necrosis factor α on megakaryopoiesis and platelet production.

BACKGROUND AND OBJECTIVES: Platelets are affected by many factors, such as infectious or aseptic inflammation, and different inflammatory states may induce either thrombocytopenia or thrombocytosis. Tumor necrosis factor α (TNFα) is an important inflammatory cytokine that has been shown to affect the activity of hematopoietic stem cells. However, its role in megakaryocyte (MK) development and platelet production remains largely unknown. This study aimed to investigate the effects of TNFα on MK and platelet generation. METHODS AND RESULTS: The ex vivo study with human CD34+ cells demonstrated that TNFα differentially modulated commitment toward the MK lineage. Specifically, a low concentration of 0.5 ng/ml TNFα promoted MK maturation, proplatelet formation, and platelet production, whereas a high concentration of 10 ng/ml or more TNFα exhibited a substantial inhibitory effect on MK and platelet production. The distinct effect of TNFα on MKs was mainly dependent on TNFα receptor 1. TNFα differentially regulated the MAPK-ERK1/2 signaling pathway and the cytoskeletal proteins cofilin and MLC2. The in vivo study with Balb/c mice indicated that low-dose or high-dose TNFα administration differentially affected short-term platelet recovery after bone marrow transplantation. CONCLUSIONS: Our study revealed distinct roles for TNFα in megakaryopoiesis and thrombopoiesis and may provide new insights regarding the treatment for platelet disorders.

Prognostic Value of Thrombocytopenia in Myelodysplastic Syndromes After Hematopoietic Stem Cell Transplantation.

Prolonged isolated thrombocytopenia (PT) is a common complication affecting the outcome of stem cell transplantation. In this study, we undertook a real-world study of 303 myelodysplastic syndrome (MDS) patients who received allogeneic hematopoietic stem cell transplantation (HSCT) between December 2007 and June 2018. 28.4% of MDS patients suffered from PT after HSCT. Survival analysis indicated that PT was associated with worse overall survival (OS) in MDS patients. The 2-year and 5-year OS in MDS patients with PT after HSCT were 49% and 47%, significantly worse than that of 68% and 60% in patients without PT (P=0.005). For RFS, patients with PT did not have an increased risk of disease relapse (P=0.964). After multivariate adjustment, PT was proved to be the independent risk factor associated with the worse OS (HR 1.49, 95% CI 1.00-2.21, P =0.048). We further analyzed risk factors associated with the occurrence of PT in MDS patients. Multiple logistic regression identified grade II-IV aGVHD, extensive chronic GVHD, hemorrhagic cystitis, and CMV activation as significant risk factors for developing PT. Among these variables, the Odds Ratio (OR) of grade II-IV aGVHD was the highest (P =0.001, OR: 2.65, 95% CI: 1.51-4.64). These data indicated the prognostic value of PT in MDS after HSCT. The identification of risk factors for PT may help improve patient management and lead to the design of effective treatment strategies.

Upregulation of HIF-1α contributes to complement activation in transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a severe complication of haematopoietic stem cell transplantation (HSCT). Complement activation is involved in the development of TA-TMA. However, the underlying mechanism is unclear. Therefore, 21 samples of TA-TMA and 1:1 matched controls were measured for hypoxia-inducible factor-1α (HIF-1α) and complement protein. The mechanism was investigated both in vitro and in vivo. In this study, we found that levels of HIF-1α were significantly higher in TA-TMA patients than that in non-TA-TMA controls. Upregulation of HIF-1α induced an increase in membrane-bound complement C3 and dysfunction of human umbilical vein endothelial cells (HUVECs) in vitro. Increasing HIF-1α in vivo led to C3 and C5b-9 deposition in the glomerular endothelial capillary complex, thrombocytopenia, anaemia, and increased serum lactate dehydrogenase (LDH) levels in wild-type (WT) but not in C3-/- mice subjected to HSCT. High platelet aggregation in peripheral blood and CD41-positive microthrombi in the kidney were also found in dimethyloxallyl glycine (DMOG)-treated mice, recapitulating the TA-TMA phenotype seen in patients. Comprehensive analysis, including DNA array, luciferase reporter assay, chromatin immunoprecipitation (ChIP)-seq, and quantitative polymerase chain reaction (PCR), revealed that HIF-1α interacted with the promoter of complement factor H (CFH) to inhibit its transcription. Decreased CFH led to complement activation in endothelial cells.

Plasma Metabolomics Identifies the Dysregulated Metabolic Profile of Primary Immune Thrombocytopenia (ITP) Based on GC-MS.

ITP is a common autoimmune bleeding disorder with elusive pathogenesis. Our study was implemented to profile the plasma metabolic alterations of patients diagnosed with ITP, aiming at exploring the potential novel biomarkers and partial mechanism of ITP. The metabolomic analysis of plasma samples was conducted using GC-MS on 98 ITP patients and 30 healthy controls (HCs). Age and gender matched samples were selected to enter the training set or test set respectively. OPLS-DA, t-test with FDR correction and ROC analyses were employed to screen out and evaluate the differential metabolites. Possible pathways were enriched based on metabolomics pathway analysis (MetPA). A total of 85 metabolites were investigated in our study and 17 differential metabolites with diagnostic potential were identified between ITP patients and HCs. MetPA showed that the metabolic disorders of ITP patients were mainly related to phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and glyoxylate and dicarboxylate metabolism. Additionally, we discriminated 6 differential metabolites and 5 enriched pathways in predicting the resistance to glucocorticoids in chronic ITP patients. The distinct metabolites discovered in our study could become novel biomarkers for the auxiliary diagnosis and prognosis prediction of ITP. Besides, the dysregulated pathways might contribute to the development of ITP.

Effectiveness and safety of leukapheresis in hyperleukocytic leukemias: a retrospective multicenter study.

Leukapheresis is an effective adjuvant therapy for leukemia patients with hyperleukocytosis, but few studies have reported recent data with modern modalities and comparisons among different leukemia types. We conducted a retrospective study on leukapheresis among 420 patients with AML, ALL and CML in four local centers. WBC counts decreased significantly (p < 0.001) postleukapheresis in all three cohorts. Clearance efficiency was higher in acute leukemia patients than CML patients (p < 0.01). Concomitant leukocytoreduction drugs improved WBC reduction only in AML patients (p < 0.05). Leukocyte, hemoglobin and platelet levels preleukapheresis might affect the clearance efficiency in AML and/or ALL patients. Hematological toxicities were the major concerns, but most of them were mild, and only 11 patients died of all causes within one week postleukapheresis. In conclusion, leukapheresis can safely reduce the leukemic burden, especially for patients with acute leukemias.

Blocking human protein C anticoagulant activity improves clotting defects of hemophilia mice expressing human protein C.

Hemophilia A and B are hereditary coagulation defects resulting in unstable blood clotting and recurrent bleeding. Current factor replacement therapies have major limitations such as the short half-life of the factors and development of inhibitors. Alternative approaches to rebalance the hemostasis by inhibiting the anticoagulant pathways have recently gained considerable interest. In this study, we tested the therapeutic potential of a monoclonal antibody, HAPC1573, that selectively blocks the anticoagulant activity of human activated protein C (APC). We generated F8-/- or F9-/- hemophilia mice expressing human protein C by genetically replacing the murine Proc gene with the human PROC. The resulting PROC+/+;F8-/- or PROC+/+;F9-/- mice had bleeding characteristics similar to their corresponding F8-/- or F9-/- mice. Pretreating the PROC+/+;F8-/- mice with HAPC1573 shortened the tail bleeding time. HAPC1573 pretreatment significantly reduced mortality and alleviated joint swelling, similar to those treated with either FVIII or FIX, of either PROC+/+;F8-/- or PROC+/+;F9-/- mice in a needle puncture-induced knee-joint bleeding model. Additionally, we found that HAPC1573 significantly improved the thrombin generation of PROC+/+;F8-/- mice but not F8-/- mice, indicating that HAPC1573 enhanced the coagulant activity of hemophilia mice by modulating human APC in vivo. We further documented that HAPC1573 inhibited the APC anticoagulant activity to improve the clotting time of human plasma deficient of FVIII, FIX, FXI, FVII, VWF, FV, or FX. These results demonstrate that selectively blocking the anticoagulant activity of human APC may be an effective therapeutic and/or prophylactic approach for bleeding disorders lacking FVIII, FIX, or other clotting factors.

CLEC-2-dependent platelet subendothelial accumulation by flow disturbance contributes to atherogenesis in mice.

Rationale: Platelets play an essential role in atherosclerosis, but the underlying mechanisms remain to be addressed. This study is to investigate the role of platelets in d-flow induced vascular inflammation and the underlying mechanism. Methods: We established a disturbed blood flow (d-flow) model by partial carotid ligation (PCL) surgery using atherosclerosis-susceptible mice and wild-type mice to observe the d-flow induced platelet accumulation in the subendothelium or in the plaque by immunostaining or transmission electron microscopy. The mechanism of platelet subendothelial accumulation was further explored by specific gene knockout mice. Results: We observed presence of platelets in atherosclerotic plaques either in the atheroprone area of aortic arch or in carotid artery with d-flow using Ldlr-/- or ApoE-/- mice on high fat diet. Immunostaining showed the subendothelial accumulation of circulating platelets by d-flow in vivo. Transmission electron microscopy demonstrated the accumulation of platelets associated with monocytes in the subendothelial spaces. The subendothelial accumulation of platelet-monocyte/macrophage aggregates reached peak values at 2 days after PCL. In examining the molecules that may mediate the platelet entry, we found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) reduced the subendothelial accumulation of platelets and monocytes/macrophages by d-flow, and ameliorated plaque formation in Ldlr-/- mice on high fat diet. Supportively, CLEC-2 deficient platelets diminished their promoting effect on the migration of mouse monocyte/macrophage cell line RAW264.7. Moreover, monocyte podoplanin (PDPN), the only ligand of CLEC-2, was upregulated by d-flow, and the myeloid-specific PDPN deletion mitigated the subendothelial accumulation of platelets and monocytes/macrophages. Conclusions: Our results reveal a new CLEC-2-dependent platelet subendothelial accumulation in response to d-flow to regulate vascular inflammation.

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.