Taisuke Kanaji - Academia.edu (original) (raw)

Papers by Taisuke Kanaji

Proceedings of the National Academy of Sciences of the United States of America, Nov 21, 2022

Infection is often associated with thrombocytopenia and rapid replenishment of platelets may be i... more Infection is often associated with thrombocytopenia and rapid replenishment of platelets may be important to maintain vascular homeostasis. We found that an activated form of tyrosyl-tRNA synthetase (YRS ACT) mimics inflammatory stress in mice, inducing a distinct population of megakaryocytes (MKs) from myeloid/MK-biased hematopoietic stem cells bypassing the classical MK-erythroid progenitor (MEP) pathway. In mice infected by lymphocytic choriomeningitis virus, myeloid/MK progenitors and MKs were mobilized from the bone marrow into the circulation and found trapped in the lung microvasculature within fibrin containing microthrombi. These findings define the role of YRS ACT in platelet generation and advance our understanding of functionally distinct alternative platelet production during viral infection.

Blood, Dec 7, 2017

Thrombin activates platelets by specific cleavage of PARs leading to platelet aggregation. Human ... more Thrombin activates platelets by specific cleavage of PARs leading to platelet aggregation. Human (h) platelets express PAR-1 and PAR-4, each with a different sensitivity to thrombin and specific kinetics of activation/deactivation influencing thrombogenesis and thrombus stability. PAR-1 and PAR-4 are candidates for the generation of anti-thrombotic drugs; indeed, a PAR-1 inhibitor (Vorapaxar) is already in clinical use. However, side effects such as bleeding warrant a continuous effort towards the generation of novel alternative inhibitors. In this regard, human PAR-4 may be a more selective antithrombotic target with a lesser impact on hemostasis. Mice are commonly used to test anti-thrombotic drugs and their mechanisms of action, but cannot be used in this instance because mouse (m) platelets express different PAR subtypes than human, i.e. PAR-3 and PAR-4. Moreover, mPAR-3 cannot be cleaved by thrombin and only acts as a co-receptor enhancing mPAR-4 response. In human platelets, PAR-1 and PAR-4 can act independently of one another. Yet, a different platelet membrane receptor, unrelated to PARs, which binds thrombin with high affinity - glycoprotein (GP) Ibα in the GPIb-IX-V complex - enhances PAR-1 response to low-dose thrombin through a mechanism only partially elucidated. Humanizing mouse platelets with respect to PAR expression would greatly facilitate explaining the functional interplay of GPIb with PAR-1 and PAR-4 and, notably, the role of these different PARs in hemostasis and thrombosis. Attempts to introduce hPAR-1 in mouse platelets have been so far unsuccessful (French et al. 2016, Arachiche et al. 2014) and claims have been made that obstacles to achieve this goal may be unsurpassable. Using a different approach, we introduced a floxed hPAR-1 transgene in the ROSA26 locus of C57BL6/J mice under the strong CAG promoter. The presence of an IRES sequence and a second transgene for eGFP allowed us to screen efficiently for platelets expressing hPAR1. Mice positive for the floxed-transgene were then bred with either a germ-line Cre recombinase-expressing strain (EIIa-Cre) or one in which Cre expression was linked to a platelet specific receptor (PF4-Cre). The percentage of platelets isolated from EIIa-Cre/PAR1+/- mice expressing eGFP was between 35 to 40% and breeding to homozygosity did not increase transgene expression. In contrast, eGFP expression was positive in nearly all PF4-Cre/PAR1+/-mice, suggesting that suppression of hPAR1 expression at the germ-line level could be advantageous for megakaryocyte development. As a functional test for transgene-expressed hPAR-1 we measured aggregation and intracellular calcium increase in washed platelets from PF4-Cre/PAR1+/+ mice stimulated by 5 μmol/liter of a specific PAR-1 activation peptide (TFLLRN, P1-AP). In both assays, the response of human or hPAR-1 expressing mouse platelets was indistinguishable; littermate control platelets lacking transgene expression (floxed hPAR1) did not respond to a 10 times higher concentration of peptide. Interestingly, when stimulated with a low dose of thrombin (0.25 nM) aggregation and intracellular calcium increase of PF4-Cre/PAR1+/+ platelets was similar to littermate controls. We reasoned that this could be a consequence of the presence of mPAR3, owning to its high affinity for thrombin. Thus we proceeded to cross PF4-Cre/PAR1+/+ with mPAR3-/- mice, so to obtain mice that express only mPAR4 and hPAR1. Aggregation induced with P1-AP (5 μM) was similar with platelets expressing PF4-Cre/PAR1+/+ and mPAR3-/-/PF4-Cre/PAR1+/+. As expected from the cofactor role of mPAR3, the minimal amount of thrombin necessary to aggregate fully platelets from mPAR3-/-/PF4-Cre/PAR1-/- was higher than in wild-type mice (2 nM vs 0.25 nM); however, 2 nM was also the minimum thrombin concentration necessary to activate mPAR3-/-/PF4-Cre/PAR1+/+ platelets. Therefore, the presence of a functional hPAR1 responsive to P1-AP does not influence the response of mouse platelets to low thrombin concentrations mediated by mPAR4, indicating that hPAR-1 and mPAR-4, like the human homologue, respond to thrombin independently of one another. In conclusion, to date we have shown that expression of a functional hPAR1 in mouse platelets is possible and should provide a versatile animal model to address several open questions on the role of platelet PAR-1 and GPIbα in hemostasis and thrombosis. Disclosures No relevant conflicts of interest to declare.

Science Translational Medicine, Mar 24, 2021

Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20–30%) des... more Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20–30%) despite modern supportive care. In a human bacteremia cohort, development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used post-myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thus providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus a “toxin-platelet-AMR” regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof-of-concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.

Blood, Nov 16, 2006

Background: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular wal... more Background: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular walls by binding to von Willebrand factor (VWF). For several decades it has been recognized that the a-subunit of GP (GPIba) also binds thrombin but the physiological relevance, if any, of this interaction was unknown. Previous studies have shown that a sulfated tyrosine 276 (Tyr276) is essential for thrombin binding to GPIba. Objectives: This study investigated the in vivo relevance of GPIba residue Tyr276 in hemostasis and thrombosis. Methods: Transgenic mouse colonies expressing the normal human GPIba subunit or a mutant human GPIba containing a Phe substitution for Tyr276 (hTg Y276F) were generated. Both colonies were bred to mice devoid of murine GPIba. Results: Surface-expressed GPIba levels and platelet counts were similar in both colonies. hTg Y276F platelets were significantly impaired in binding a-thrombin but displayed normal binding to type I fibrillar collagen and human VWF in the presence of ristocetin. In vivo thrombus formation as a result of chemical damage (FeCl 3) demonstrated that hTg Y276F mice have a delayed time to occlusion followed by unstable blood flow indicative of embolization. In models of laser-induced injury, thrombi developing in hTg Y276F animals were also less stable. Conclusions: The results demonstrate that GPIba residue Tyr276 is physiologically important, supporting stable thrombus formation in vivo.

Japanese Journal of Thrombosis and Hemostasis, 2014

Blood, Nov 16, 2004

The hereditary bleeding disorder Bernard-Soulier syndrome (BSS) is caused by an absent or dysfunc... more The hereditary bleeding disorder Bernard-Soulier syndrome (BSS) is caused by an absent or dysfunctional GPIb/IX complex. Lopez et al. have reported that three subunits of the complex (GPIbα, GPIbβ and GPIX) are all necessary for efficient translocation to the cell surface. In contrast, Meyer et al. have demonstrated that GPIbα alone can incorporate into the cell surface. We have established a cell culture system with inducible levels of GPIbβ and GPIX. Using these cell lines, we characterize an intrinsic ability of GPIbα to form dimers and the relevance of specific GPIbα sequences for dimer formation and the assembly of a GPIb/IX complex. A stable CHO cell line was established that expresses GPIbβ and GPIX under the control of Tet-responsive elements. This cell line was then transfected with WT GPIbα and truncated GPIbα mutants. Expression was monitored by Western blot analysis and flow cytometry. We observed that expression of GPIbα, by itself, targets GPIbα to the cell surface as a dimer albeit at reduced levels. The GPIbα binding domain for filamin (residues 545–605) is essential for forming a dimer but not for assembly of a GPIb/IX complex. We also observed GPIbα dimer formation in a transgenic mouse that expresses human GPIbα and a mouse chimeric GPIb-IX complex suggesting dimer formation can occur in the platelet. To investigate the role of filamin in the dimerization process, we established a stable cell line deficient in filamin by targeting filamin expression with RNAi. We observed GPIbα dimers in the absence of filamin expression. These results illustrate the importance of expressing all three subunits of the GPIb/IX complex, but also document as intrinsic potential within GPIbα for surface expression via intermolecular homodimerization.

Blood, 2021

Desialylation has been recognized as a mechanism for platelet clearance in various conditions, in... more Desialylation has been recognized as a mechanism for platelet clearance in various conditions, including infection and immune thrombocytopenia (ITP), and plays a role in the removal of aged platelets. However, it remains unclear whether, and if so how, MK desialylation affects thrombopoiesis. The study by Lee-Sundlov et al identified a novel surveillance system of MK sialylation status by pDC-like immune cells, leading to inhibition of platelet production from MKs. Targeted deletion of O-glycan sialyltransferase (St3gal1), specifically in MK lineage (St3gal1), generated a mouse model with increased ThomsenFriedenreich (TF) antigen expression on MKs. TF antigen, which is normally masked by terminal sialylation, becomes exposed when St3gal1 is deleted. The St3gal1 mice had thrombocytopenia with platelet counts at 50% of the control mice. Interestingly, thrombocytopenia in St3gal1 mice was reversed by treatment with dexamethasone or targeted deletion of Jak3, suggesting an immune-media...

Thrombosis Research, 2022

Blood, 2010

2022 Introduction: Sitosterolemia is a rare, autosomal recessive disorder characterized by the ac... more 2022 Introduction: Sitosterolemia is a rare, autosomal recessive disorder characterized by the accumulation of plant sterols in blood and tissues, and is caused by mutations in one of the adenosine triphosphate-binding cassette (ABC) transporter ABCG5 or ABCG8 genes. Patients with mutations in either of these sterol transport proteins, which normally form a heterodimer sterol egress channel, frequently develop tendon and cutaneous xanthomas and, most importantly, are at risk of developing premature coronary atherosclerosis. Other clinical manifestations include hematological abnormalities such as hemolytic anemia, macrothrombocytopenia, and loss of ristocetin-induced platelet agglutination – a measure of the ability of platelet glycoprotein (GP) Ib to function as a adhesion receptor for von Willebrand factor (VWF). Mice genetically deficient in ABCG5 or ABCG8 fully recapitulate the macrothrombocytopenia and loss of platelet function seen in human sitosterolemia, a condition that can...

Thrombosis and Haemostasis, 2001

SummaryThe factor XII genes of two unrelated factor XII-deficient Japanese families were screened... more SummaryThe factor XII genes of two unrelated factor XII-deficient Japanese families were screened, and two novel mutations were identified. A heterozygous mutation (Q421K) was identified in the gene of a cross-reacting material (CRM)-negative patient with reduced FXII activity (entitled Case 1). No mutations were discovered in the other allele. Case 2 was a CRM-negative patient with severe FXII deficiency. In this case, a homozygous mutation (R123P) was discerned. Expression studies in Chinese Hamster Ovary (CHO) cells demonstrated accumulation of mutant Q421K factor XII in the cell, and insufficient secretion, while the R123P mutant showed lower levels of accumulation than wild-type, and no evidence of secretion in culture supernatant. In the presence of proteasome inhibitor, all types of FXII (wild-type, Q421K, R123P) accumulated in the cells. Protease protection experiments using the microsomal fraction of these cell lines demonstrated that while 20% wild-type FXII (total wild-ty...

Trends in Biochemical Sciences

Blood, 2006

To resolve the lack of sources for products used in blood transfusion therapy, the utilization of... more To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry wi...

Blood, 2016

Introduction: The interaction between von Willebrand Factor (VWF) and platelet glycoprotein (GP) ... more Introduction: The interaction between von Willebrand Factor (VWF) and platelet glycoprotein (GP) Ibα is key for initiating the response to vascular injury that leads to hemostasis or, in pathological conditions, may be a cause of thrombosis. VWF binding to GPIbα occurs through the A1 domain (VWFA1) and its role in platelet adhesion and aggregation becomes progressively more important with increasing shear rates, i.e., in arterioles or pathologically stenosed arteries. Owing to the key role in platelet adhesion/aggregation under arterial flow conditions, VWFA1 has been considered an obvious target for antithrombotic intervention. However, efforts to develop this concept have been complicated by the lack of suitable animal models due to species-specificity in VWFA1-GPIb binding. To obviate the problem, we have generated new mouse strains with humanized VWF-GPIb interaction and characterized the resulting phenotypes in experimental ex vivo and in vivo models of hemostasis and thrombosi...