BACKGROUND:Integrin β3 orchestrates thrombotic and inflammatory processes through bidirectional signaling, with Src acting as a key downstream effector. OBJECTIVES:To dissect this axis, we generated SrcE97A mice that selectively disrupt the interaction between Src and integrin β3, impairing outside-in signaling while preserving inside-out signaling through integrin β3. METHODS:Platelet function was assessed through clot retraction, irreversible aggregation, thrombus stabilization under flow, and soluble fibrinogen binding. Thrombosis models included FeCl3-induced carotid artery and laser-induced cremaster arteriole injury for arterial thrombosis, and the inferior vena cava ligation for deep vein thrombosis. Hemostasis was evaluated in parallel. The impact on cerebral infarction was measured in vivo. In addition, the biological functions of αvβ3 outside-in signaling were also analyzed. RESULTS:The SrcE97A mutation impaired outside-in signaling while preserving inside-out signaling in platelets. This suppressed αIIbβ3-mediated clot retraction, secondary aggregation, and thrombus stabilization under flow. SrcE97A mice showed reduced arterial and venous thrombosis without hemostatic compromise. These antithrombotic effects were reinforced by protection against cerebral infarction in vivo. Furthermore, the E97A mutation influenced immunologic regulation. Specifically, it promoted pulmonary inflammation by skewing T helper (Th) cell differentiation toward Th1, via disruption of the αvβ3-Src-STAT4/6 signaling axis. CONCLUSIONS:These findings establish integrin β3 as a dual regulator of thrombosis and immune homeostasis. Selective disruption of outside-in signaling attenuates pathologic thrombosis without compromising hemostasis and reshapes Th1/Th2 balance. Targeting the Src E97 interface may offer strategies to inhibit pathologic thrombosis and modulate inflammation.

Glanzmann thrombasthenia (GT) is an inherited hemorrhagic disorder characterised by impaired platelet functions, manifested clinically as spontaneous bleeding. It is usually inherited in an autosomal recessive manner. Platelet dysfunction in patients with GT is caused by quantitative and/or qualitative deficiencies in αIIbβ3, which result from mutations in the genes encoding αIIbβ3. These genetic alterations lead to platelet dysfunction characterised by impaired fibrinogen binding capacity upon agonist stimulation, defective aggregation and spreading. While classical GT typically exhibits normal platelet counts and morphology, very rare mutations in ITGA2B (encoding αIIb) and/or ITGB3 (encoding β3) cause macrothrombocytopenia or increased platelet anisotropy (heterogeneity of platelet size and morphology). This type of mutation mainly localises in the membrane-proximal region of αIIbβ3 and is inherited in an autosomal dominant manner. This particular type of disorder is called ITGA2B/ITGB3-related macrothrombocytopenia and has been considered a subset of congenital macrothrombocytopenia. Current research suggests that gain-of-function mutations in ITGA2B or ITGB3 underlie the pathogenesis of most ITGA2B/ITGB3-related macrothrombocytopenia and mechanistically distinguish it from classical GT. However, recent reports have documented non-activating ITGB3 mutations that also cause macrothrombocytopenia, presenting a profound challenge to the mechanistic understanding of ITGA2B/ITGB3-related macrothrombocytopenia. This review summarises the reported cases of gain-of-function mutations in ITGA2B and ITGB3 associated with ITGA2B/ITGB3-related macrothrombocytopenia hitherto and discusses the potential molecular pathways contributing to the unique phenotypes in ITGA2B/ITGB3-related macrothrombocytopenia.

Background:Pulmonary immune homeostasis requires tight control of adaptive responses. Integrin β3 is a well-known mediator of cell adhesion and platelet function. However, its role in adaptive immunity, especially in B cell responses, remains unclear. Methods:We defined the pulmonary phenotype of constitutive β3-deficient (β3-/-) mice by histopathology. We performed integrated transcriptomic and proteomic profiling of lung tissue to map the molecular signature of spontaneous pulmonary inflammation. We further probed the underlying mechanisms with additional histology and functional assays and tested for biological significance using transcriptomics data from auto-immune disease patients. Results:β3-/- mice developed spontaneous pulmonary inflammation marked by B cell activation and in situ immune-complex deposition within alveoli. Multi-omics integration implicated the CD40-CD40 Ligand (CD40L) axis as a central driver of this pathology. Mechanistically, loss of β3 enhanced CD40L-CD40 engagement on B cells, resulting in NF-κB pathway hyperactivation. Consistent with our murine data, reduced ITGB3 expression in patients with autoimmune disease correlated with transcriptional signatures of B cell activation and inflammation. Conclusions:These results reframe integrin β3 as a threshold regulator of B cell activation. The β3-CD40L-CD40 axis therefore represents a potential therapeutic target for B cell-mediated autoimmune diseases.

BACKGROUND:RIP2 (receptor-interacting protein kinase 2) is an essential mediator of inflammation and innate immunity downstream of PRRs (pattern recognition receptors). Platelets express RIP2, while its role in platelet activation, thrombosis, and myocardial infarction, and whether these effects are mediated through the PRR pathway, is unknown. METHODS:In vitro assays of platelet aggregation, dense and α-granule secretion, spreading, and clot retraction, along with an ex vivo microfluidic whole blood perfusion assay, and in vivo models of FeCl3-induced mesenteric arteriolar thrombosis and ischemia/reperfusion myocardial infarction, were used to assess the impact of RIP2 deficiency on platelet function. Immunoprecipitation followed by liquid chromatography-tandem mass spectrometry was performed to elucidate the mechanism by which RIP2 limits dense granule secretion in platelets. Furthermore, RIP2 expression levels in platelets from healthy donors and patients with coronary artery disease were measured by Western blotting to evaluate their clinical relevance. RESULTS:Here, we show that RIP2 deficiency enhances platelet dense granule secretion in response to GPIb (platelet glycoprotein Ib) and GPVI (platelet glycoprotein VI) activation; platelet aggregation in whole blood and adhesion under arterial shear are also increased. Consistently, RIP2 inhibitor WEHI-345 potentiates human platelet dense granule secretion and inhibits RIP2 phosphorylation induced by thrombin and collagen. These phenotypes are translated into shorter bleeding time and accelerated FeCl3-induced arterial thrombosis. Importantly, platelets from patients with coronary artery disease and mice with atherosclerosis express lower RIP2, and RIP2 deficiency worsens myocardial infarction and cardiac function in a mouse ischemia/reperfusion model. Mechanistically, we found that platelets express DOCK8 (dedicator of cytogenesis protein 8), which is sequestered by p-RIP2 (phosphorylated RIP2), causing inhibition of Cdc42 (cell division cycle 42) activation and subsequent dense granule release. CONCLUSIONS:RIP2 restrains platelet activation and thrombosis, thereby mitigating myocardial infarction. We identify a novel PRR-independent pathway, p-RIP2-DOCK8-Cdc42, which suppresses dense granule release downstream of GPIb and GPVI. Targeting the platelet RIP2 pathway may offer a therapeutic strategy against atherothrombotic diseases from early atherosclerosis to arterial thrombosis and myocardial infarction.

Platelet spreading and clot retraction, albeit both mediated by integrin outside-in signaling, lead to platelet shape changes in two opposite directions. The mechanisms by which these processes are regulated are not fully understood. Our previous study found that E726Q mutation in β3 integrin caused impaired spreading in Chinese hamster ovary (CHO) cells on immobilized fibrinogen.The current study further utilized knock-in mice bearing the β3E726Q mutation to explore the underlying mechanisms whereby the E726 residue differentially influences platelet spreading and clot retraction.Compared to wild type (WT) platelets, β3E726Q platelets displayed similar level of β3 expression but partially impaired fibrinogen binding associated with attenuated responses in platelet aggregation and P-selectin exposure. Notably, β3E726Q mutation resulted in defective platelet spreading but accelerated clot retraction concomitant with increased clot density. Functionally, β3E726Q mice displayed prolonged bleeding time and defective thrombogenesis in vitro and in vivo. Further mechanistic study showed that in β3E726Q platelets the activities of RhoA and Rac1 were significantly enhanced following thrombin stimulation, possibly due to reduced binding of Gα13 to the β3 cytoplasmic tail.Taken together, the β3E726 is a potential novel regulatory site that influences the direct interaction of β3 cytoplasmic tail with Gα13 and therefore the activity of downstream RhoA, a molecular switch that shifts platelet spreading into clot retraction.

Objective Receptor-interacting protein 2 (RIP2) is an essential mediator of inflammation and innate immunity downstream of pattern recognition receptors (PRRs). Platelets express RIP2, while its role in platelet activation, thrombosis, and myocardial infarction (MI) is unknown. Approach and Results Here we show that RIP2 deficiency enhances platelet dense granule secretion in response to GPIb and GPVI activation; platelet aggregation in whole blood and adhesion under arterial shear are also increased. Consistently, RIP2 inhibitor WEHI-345 potentiates human platelet dense granule secretion and inhibits RIP2 phosphorylation induced by thrombin and collagen. These phenotypes are translated into shorter bleeding time, accelerated FeCl3-induced arterial thrombosis. Importantly, platelets from patients with coronary artery disease and mice with atherosclerosis express lower RIP2, and RIP2 deficiency deteriorates MI and cardiac function in a mouse ischemia/reperfusion model. Mechanistically, we found that platelets express dedicator of cytogenesis protein 8 (DOCK8), which is sequestered by phosphorylated RIP2 (p-RIP2), causing inhibition of Cdc42 activation and subsequent dense granule release. Conclusions In conclusion, RIP2 inhibits platelet activation, thrombosis, and ameliorates MI. Our results also suggest that a novel PRR-independent pathway, p-RIP2-DOCK8-Cdc42, negatively regulates platelet activation downstream of GPIb and GPVI. Platelet RIP2 pathway may be promising for therapeutic intervention of atherothrombotic diseases from early atherosclerosis stage to arterial thrombosis and MI. Highlights ### Competing Interest Statement The authors have declared no competing interest. * RIP2 : Receptor-interacting protein 2 PRRs : pattern recognition receptors MI : myocardial infarction GPIb : Platelet glycoprotein Ib GPVI : Platelet glycoprotein VI DOCK8 : dedicator of cytogenesis protein 8 Cdc42 : cell division cycle 42 TXA2 : thromboxane A2 vWF : von Willebrand factor NOD2 : Nucleotide-binding oligomerization domain 2 CARD : Caspase Recruitment Domain CRP : collagen related peptide CAD : coronary heart disease

Background:Antiplatelet and anticoagulation are the cornerstones for arterial and venous thrombosis, respectively; however, hemorrhage remains a significant clinical challenge. Platelets are crucial for arterial thrombosis and contribute to venous thrombosis. Integrin β3 mediates outside-in signaling, which is critical for thrombosis, while inside-out signaling maintains hemostasis. Targeting the β3/Src interactions to selectively inhibit outside-in signaling offers a promising antithrombotic strategy without compromising hemostasis. Objectives:To develop more potent small molecules that selectively disrupt the β3/Src interaction, thereby inhibiting arterial and venous thrombogenesis without increasing bleeding risk. Methods:Building on the previously identified compound DCDBS84, we developed the structurally modified small molecules C109 and C116, with enhanced affinity for the Src SH3 domain. Their antithrombotic effects on both arterial and venous thrombosis were systematically evaluated through in vitro and in vivo studies. The impact on hemostatic function was assessed using a tail-bleeding model. Additionally, the drug developability of C109 was assessed via pharmacokinetic (PK) and metabolite analysis. Results:C109 and C116 exhibited superior efficacy in disrupting the β3/Src interaction. In vitro and in vivo studies demonstrated that C109 and C116 effectively suppress thrombosis at levels comparable to high doses of the αIIbβ3 antagonist integrilin, without elevating bleeding risk. In the Stenosis Model, C109 and C116 significantly reduced venous thrombogenesis by suppressing platelet activation and neutrophil extracellular trap formation. Additionally, C109 displayed favorable PK properties and robust metabolic stability. Conclusion:These findings identify promising small molecules that inhibit thrombosis while maintaining hemostasis, providing new avenues for safer and more effective clinical management.

Venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE), is a complex vascular disorder with high morbidity and mortality, driven by Virchow's Triad: blood stasis, hypercoagulability, and endothelial injury. VTE is now recognized as an inflammatory process involving multiple components. Platelets are involved in the process of VTE, contributing to thrombosis initiation, progression, resolution and recurrence through coagulation activation, and interactions with immune and endothelial cells. Anticoagulation remains the cornerstone of VTE treatment; however, antiplatelet agents like aspirin have demonstrated therapeutic potential, particularly following major orthopedic surgeries. Furthermore, emerging platelet-targeted therapies and biomarkers offer new opportunities for improving VTE diagnosis and treatment. This review explores the evolving role of platelets in VTE pathophysiology, assesses current antiplatelet strategies, and highlights novel therapeutic approaches. Advancing platelet research in VTE may lead to safer, more effective interventions, optimizing outcomes for patients with this life-threatening condition.

Aims Dedicator of cytokinesis 2 (DOCK2), a member of the DOCK family of guanine nucleotide exchange factors that specifically act on the Rho GTPases including Rac and Cdc42, plays pivotal roles in the regulation of leukocyte homeostasis. However, its functions in platelets remain unknown.Methods and results Using mice with genetic deficiency of DOCK2 (Dock2-/-), we showed that Dock2-/- mice exhibited a macrothrombocytopenic phenotype characterized as decreased platelet count and enlarged platelet size by transmission electron microscopy. Dock2-/- megakaryocytes had reduced polyploidization determined by propidium iodide staining and defective proplatelet formation by confocal microscopy. DOCK2 deficiency led to enriched F-actin level in resting platelets but defective F-actin assembly in activated platelets by phalloidin staining, and mechanistically, attenuated activity of Rac1, unchanged Cdc42 but enhanced RhoA measured by immunoprecipitation of GTP-bound proteins. Immunoblotting analysis showed that Dock2-/- platelets had reduced immunoreceptor tyrosine-based activation motif signaling downstream of impaired clustering of GPVI receptors determined by stochastic optical reconstruction microscopy. Further, DOCK2 deficiency resulted in reduced density and branches of fibrin fibres in the clots in vitro and diminished platelet aggregation in a microfluidic chamber ex vivo. Dock2-/- platelets exhibited impaired incorporation into a growing thrombus in cremaster arterioles following allogeneic transfusion into a WT recipient and defective heterotypic interactions with neutrophils in cremaster venules as reflected by decreased platelet-neutrophil aggregate formation in vitro under stirring condition. In addition, myeloid deficiency of DOCK2 caused prolonged tail bleeding times. Finally, pharmacological inhibition of DOCK2 using a small-molecular inhibitor CPYPP suppressed actin dynamics leading to impaired responses to GPVI activation and defects in platelet spreading, clot retraction, and thrombus formation.Conclusion DOCK2 plays critical roles in the regulation of platelet biogenesis and functions by controlling Rac1 activity and cytoskeletal actin dynamics and may be a novel target for the treatment of thrombotic and thrombo-inflammatory diseases.

Background Tryptanthrin (Couroupitine A) is isolated from indigo-bearing traditional Chinese herbal medicines. It has a broad spectrum of pharmacological and biological activities. However, the potential effects of tryptanthrin on platelet function and thrombus formation remain elusive. Methods Platelets were harvested from C57BL/6 mice and healthy individuals. Following incubation with tryptanthrin, various platelet functions were assessed. Thrombus formation in the presence of tryptanthrin was evaluated both in vitro, using a BioFlux 200 microfluidic system, and in vivo, through FeCl3-induced thrombosis and mouse deep venous thrombosis experiments. The closure times of the tryptanthrin-treated whole blood samples were determined using the PFA-200 system. Platelet proteomics sequencing was conducted to elucidate the underlying mechanisms by which tryptanthrin influences platelet function. Results Tryptanthrin inhibited mouse platelet function and impaired carotid artery and deep venous thrombus formation. Tryptanthrin also inhibited human platelet spreading, aggregation and clot retraction. The signaling pathways related to platelet activation, aggregation, hemostasis, and the fibrin clotting cascade were significantly suppressed in platelets treated with tryptanthrin. Notably, the expression of Gp1bα in platelets was diminished by tryptanthrin. Conclusions Tryptanthrin impairs platelet function and thrombus formation.

Although immunotherapy is expanding treatment options for cancer patients, the prognosis of advanced cancer remains poor, and these patients must contend with both cancers and cancer-related thrombotic events. In particular, immune checkpoint inhibitors are associated with an increased risk of atherosclerotic thrombotic events. Given the fundamental role of platelets in atherothrombosis, co-administration of antiplatelet agents is always indicated. Platelets are also involved in all steps of cancer progression. Classical antithrombotic drugs can cause inevitable hemorrhagic side effects due to blocking integrin β3 bidirectional signaling, which regulates simultaneously thrombosis and hemostasis. Meanwhile, many promising new targets are emerging with minimal bleeding risk and desirable anti-tumor effects. This review will focus on the issue of thrombosis during immune checkpoint inhibitor treatment and the role of platelet activation in cancer progression as well as explore the mechanisms by which novel antiplatelet therapies may exert both antithrombotic and antitumor effects without excessive bleeding risk.

Corona virus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has affected the whole world. Acquired thrombotic thrombocytopenic purpura (TTP) has been reported after administration of mRNA- or adenoviral vector-based COVID-19 vaccines, including Ad26.COV2-S, BNT162b2, mRNA-1273, and ChAdOx1 nCov-19. However, whether inactivated vaccines, such as CoronaVac, could cause TTP and whether the symptoms in TTPs caused by inactivated vaccines are different from previously reported cases are unknown. In this study, two cases were reported. Both cases developed TTP after the second CoronaVac vaccination shot, but not the first. They demonstrated symptoms of fever, neurological abnormalities, renal dysfunction, thrombocytopenia, and hemolysis. Both patients achieved complete remission through several sessions of plasma exchanges and immune suppression. The incidence of TTP in Nanjing area was analyzed. The number of patients with TTP was 12 in 2019, 6 in 2020, 16 in 2021, and 19 in 2022. To the authors’ knowledge, this report is the first report of TTP associated with inactivated COVID-19 vaccine (CoronaVac). The rarity and delayed onset may be due to the relatively milder immune response caused by the inactivated vaccines than mRNA-based ones. Timely plasma exchange is a vital treatment for CoronaVac-related TTP, similar to activated vaccine-related TTP.

Integrins are heterodimeric receptors comprising α and β subunits. They are expressed on the cell surface and play key roles in cell adhesion, migration, and growth. Several types of integrins are expressed on the platelets, including αvβ3, αIIbβ3, α2β1, α5β1, and α6β1. Among these, physically αIIbβ3 is exclusively expressed on the platelet surface and their precursor cells, megakaryocytes. αIIbβ3 adopts at least three conformations: i) bent-closed, ii) extended-closed, and iii) extended–open. The transition from conformation i) to iii) occurs when αIIbβ3 is activated by stimulants. Conformation iii) possesses a high ligand affinity, which triggers integrin clustering and platelet aggregation. Platelets are indispensable for maintaining vascular system integrity and preventing bleeding. However, excessive platelet activation can result in myocardial infarction (MI) and stroke. Therefore, finding a novel strategy to stop bleeding without accelerating the risk of thrombosis is important. Regulation of αIIbβ3 activation is vital for this strategy. There are a large number of molecules that facilitate or inhibit αIIbβ3 activation. The interference of these molecules can accurately control the balance between hemostasis and thrombosis. This review describes the structure and signal transduction of αIIbβ3, summarizes the molecules that directly or indirectly affect integrin αIIbβ3 activation, and discusses some novel antiαIIbβ3 drugs. This will advance our understanding of the activation of αIIbβ3 and its essential role in platelet function and tumor development.

To clarify whether arsenic could exert inhibitory effects on tumor cells in pleural effusions of patients with non-small cell lung cancer (NSCLC), 36 NSCLC pleural effusion samples were collected from Changzheng Hospital and Ruijin Hospital, from 2019 to 2022. The genotype of epidermal growth factor receptor (EGFR) was identified. Tumor cells were isolated and treated with arsenic trioxide (ATO) or/and gefitinib. Additionally, six patients were intrapleurally administrated with ATO. Results showed that 25 samples bore EGFR wild type (WT) and 11 harbored EGFR mutations, including 6 with L858R, 3 with ΔE746-A750, and 2 with T790M. ATO diminished the number of tumor cells from patients with WT and mutant EGFR, down-regulated the expression or phosphorylation of EGFR, pmTOR, PI3K, PTEN, and p4E-BP1, and up-regulated the expression of LC3. Immunofluorescent experiments showed that ATO enhanced LC3 and P62. By contrast, gefitinib was only effective in those harboring EGFR sensitizing mutations. Notably, in patients with intrapleural ATO injection, the pleural effusion underwent a bloody to pale yellow color change, the volume of the pleural effusion was reduced, and the number of the tumor cells was significantly reduced. In conclusion, arsenic is effective against NSCLC with various EGFR genotypes in vitro and in vivo, and potentially circumvents gefitinib resistance.

In acute promyelocytic leukemia (APL), characterized by the promyelocytic leuke-mia/retinoic acid receptor a (PML-RARa) fusion protein, hemorrhage is the most serious and intractable problem and is the leading cause of the early death and the treatment failure in APL. Platelet anomalies, coagulopathy, hyperfibrinolysis, and disseminated intravascular coa-gulation (DIC) are implicated in the bleeding diathesis in APL in which PML-RARa-mediated aberrant expression of tissue factor (TF) plays a critical role. This review discusses the patho-logical processes that contribute to the bleeding diathesis in APL and their ways to regulate hemostasis, coagulation and fibrinolysis with a focus on the regulatory mechanisms of eleva-ted TF expression in APL, the transcriptional regulation of TF at promoter region that leads to the aberrant expression of TF, and the inhibitory effects of ATRA and arsenic trioxide (ATO) on TF expression and function. In sum, dysregulated coagulatory and hemostatic mechanisms are responsible for the bleeding diathesis of APL, and the PML-RARa/TF/coagulopathy/DIC axis deserves much attention. Deeper understanding of these mechanisms would help to develop novel targeting strategies for combating the hemorrhagic death of APL patients. (c) 2023 Published by Elsevier Masson SAS on behalf of l'Academie nationale de medecine.

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC + rLC protein had a higher activity than the hHC + hLC protein at comparable expression levels. The specific activity of hHC + rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC + rLC treatment exhibited a visibly higher activity than those treated with hHC + hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC + rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.

The use of messenger RNA (mRNA) enables the transient pro-duction of therapeutic proteins with stable and predictable translational kinetics and without the risk of insertional muta-genesis. Recent findings highlight the enormous potential of mRNA-based therapeutics. Here, we describe the synthesis of chemically modified thrombopoietin (TPO) mRNA through in vitro transcription and in vivo delivery via lipid nanopar-ticles (LNPs). After delivery of TPO mRNA in mice, compared with normal physiological values, plasma TPO protein levels increased over 1000-fold in a dose-dependent manner. More-over, through a single intravenous dose of TPO mRNA-loaded LNPs, both reticulated and total platelet count increased signif-icantly in mice, demonstrating that TPO protein derived from the exogenous mRNA was able to maintain normal activity. Submicrogram quantity of N1-methylpseudouridine-modified TPO mRNA showed a similar effect in promoting thrombopoi-esis as that by the TPO receptor agonist romiplostim. In addition, a therapeutic value was established in anti-GPIba (CD42b) antibody-induced thrombocytopenia mouse models that showed a fast recovery of platelet count. Our study demon-strated chemically modified in-vitro-transcribed TPO mRNA as a potentially safe therapeutic intervention to stimulate thrombopoiesis.

Conventional antiplatelet agents indiscriminately inhibit both thrombosis and hemostasis, and the increased bleeding risk thus hampers their use at more aggressive dosages to achieve adequate effect. Blocking integrin α IIb β 3 outside-in signaling by separating the β 3/Src interaction, yet to be proven in vivo, may nonetheless resolve this dilemma. Identification of a specific druggable target for this strategy remains a fundamental challenge as Src SH3 is known to be responsible for binding to not only integrin β 3 but also the proteins containing the PXXP motif. In vitro and in vivo mutational analyses show that the residues, especially E97, in the RT loop of Src SH3 are critical for interacting with β 3. DCDBS84, a small molecule resulting from structure-based virtual screening, is structurally validated to be directed toward the projected target. It specifically disrupts β 3/Src interaction without affecting canonical PXXP binding and thus inhibits the outside-in signaling-regulated platelet functions. Treatment of mice with DCDBS84 causes a profound inhibition of thrombosis, equivalent to that induced by extremely high doses of α IIb β 3 antagonist, but does not compromise primary hemostasis. Specific targets are revealed for a preferential inhibition of thrombosis that may lead to new classes of potent antithrombotics without hemorrhagic side effects.

OBJECTIVE To explore the effects of the rat FVIII light chain (rLC) on the activity of human FVIII heavy chain hHC745 and hHC1690. METHODS hHC745, hHC1690, human FVIII light chain (hLC) and rLC were cloned into adeno-associated virus serotype 8 (AAV8) expression vectors with CB promoter (ubiquitous expression), respectively, and transfected into 293T cells using a dual-chain strategy of co-expression of HC and LC. The cultured cell supernatant was collected at 48 hours after transfection. The plasmids (pAAV8-CB-hHC745, pAAV8-CB-hHC1690, pAAV8-CB-hLC and pAAV8-CB-rLC) were hydrodynamically injected into hemophilia A (HA) mice via lateral tail vein. Forty-eight hours after injection, the peripheral blood of the mice was collected through retroorbital venous plexus and the plasma was obtained by centrifugation. The activity of FVIII was detected by activated partial thromboplastin time (aPTT) assay, and the antigen expression level of FVIII was determined by enzyme-linked immunosorbent assay (ELISA). The specific activity of FVIII was calculated based on the activity and the antigen expression level. RESULTS In 293T cells, the coagulation activity of FVIII was significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC. The FVIII activity of hHC745+rLC was about 4.6 times higher than that of hHC745+hLC, while hHC1690+rLC was about 2.9 times higher than that of hHC1690+hLC. The data from ELISA showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.1 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 2.8 times compared with hHC1690+hLC. In HA mice administrated with hydrodynamic injection, the FVIII activity of hHC745+rLC and hHC1960+rLC was significantly higher than that of hHC745+hLC and hHC1690+hLC at comparable expression level. The FVIII activity of hHC745+rLC was significantly higher than that of hHC745+hLC, increasing to about 5.1 times, while, that of hHC1690+rLC increasing to about 2.1 times than hHC1690+hLC. ELISA results also showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.2 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 1.8 times compared with hHC1690+hLC. In addition, the activity of hHC1690 combined with rLC was significantly higher than that of hHC745 combined with rLC. CONCLUSION rLC can significantly enhance the coagulation activity of FVIII when co-expressed with hHC of different length including hHC745 and hHC1690.

Abstract Background Treatment of hemophiliacs with inhibitors remains challenging, and new treatments are in urgent need. Coagulation factor X plays a critical role in the downstream of blood coagulation cascade, which could serve as a bypassing agent for hemophilia therapy. Base on platelet‐targeted gene therapy for hemophilia by our and other groups, we hypothesized that activated factor X (FXa) targeted stored in platelets might be effective in treating hemophilia A (HA) and B (HB) with or without inhibitors. Methods To achieve the storage of FXa in platelets, we constructed a FXa precursor and used the integrin αIIb promoter to control the targeted expression of FXa precursor in platelets. The expression cassette (2bFXa) was carried by lentivirus and introduced into mouse hematopoietic stem and progenitor cells (HSPCs), which were then transplanted into HA and HB mice. FXa expression and storage in platelets was examined in vitro and in vivo. We evaluated the therapeutic efficacy of platelet‐stored FXa by tail bleeding assays and the thrombelastography. In addition, thrombotic risk was assessed in the recipient mice and the lipopolysaccharide induced inflammation mice. Results By transplanting 2bFXa lentivirus‐transduced HSPCs into HA and HB mice, FXa was observed stably stored in platelet α‐granules, the stored FXa is releasable and functional upon platelet activation. The platelet‐stored FXa can significantly ameliorate bleeding phenotype in HA and HB mice as well as the mice with inhibitors. Meanwhile, no FXa leakage in plasma and no signs of increased risk of hypercoagulability were found in transplantation recipients and lipopolysaccharide induced septicemia recipients. Conclusions Our proof‐of‐principle data indicated that target expression of the FXa precursor to platelets can generate a storage pool of FXa in platelet α‐granules, the platelet‐stored FXa is effective in treating HA and HB with inhibitors, suggesting that this could be a novel choice for hemophilia patients with inhibitors.