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1.
Am J Hematol ; 95(8): 960-965, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32356313

RESUMO

With licensure of extended half-life (EHL) factor products and the changing landscape of available hemophilia products, patients and providers have options for less treatment-intense prophylaxis. The impact of these products in clinical practice to date remains understudied. We aimed to quantify the use of EHL products in prophylaxis in the US using the ATHN-dataset, a database of 145 ATHN-affiliated hemophilia treatment centers (HTCs). Further, we aimed to quantify the impact of EHL on key hemophilia indicators including annualized bleed rates (ABRs), hemophilia joint health scores (HJHS) and quality of life (QOL) metrics. The use of EHL vs standard half-life (SHL) products in severe hemophilia was compared between June 2018 and March 2019 using the ATHN-dataset. A cohort of patients was also recruited from seven participating HTCs in order to compare ABR, HJHS and QOL between product classes. By March 2019 the number of individuals with severe Hemophilia A (SHA) receiving EHLs remained relatively stable (28.4%), whereas the number of prescribed non-factor products increased to 7.1%, with a diminishing majority of patients (64.0%) continuing to receive SHLs. The majority of patients with severe hemophilia B (SHB) received treatment with EHLs including 57.5% by March 2019. There was a trend toward lower ABR with use of EHLs in SHA and SHB, although this did not result in improved HJHS nor QOL. EHL use in the United States in severe hemophilia continues to increase, although at a slower rate in SHA with the availability of non-factor therapy. The impact of the EHL therapies in clinical practice should continue to be examined prospectively.


Assuntos
Meia-Vida , Hemofilia A/terapia , Adolescente , Adulto , Idoso , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estados Unidos , Adulto Jovem
2.
J Cell Sci ; 130(21): 3764-3775, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28954813

RESUMO

Kindlins play an important role in supporting integrin activation by cooperating with talin; however, the mechanistic details remain unclear. Here, we show that kindlins interacted directly with paxillin and that this interaction could support integrin αIIbß3 activation. An exposed loop in the N-terminal F0 subdomain of kindlins was involved in mediating the interaction. Disruption of kindlin binding to paxillin by structure-based mutations significantly impaired the function of kindlins in supporting integrin αIIbß3 activation. Both kindlin and talin were required for paxillin to enhance integrin activation. Interestingly, a direct interaction between paxillin and the talin head domain was also detectable. Mechanistically, paxillin, together with kindlin, was able to promote the binding of the talin head domain to integrin, suggesting that paxillin complexes with kindlin and talin to strengthen integrin activation. Specifically, we observed that crosstalk between kindlin-3 and the paxillin family in mouse platelets was involved in supporting integrin αIIbß3 activation and in vivo platelet thrombus formation. Taken together, our findings uncover a novel mechanism by which kindlin supports integrin αIIbß3 activation, which might be beneficial for developing safer anti-thrombotic therapies.


Assuntos
Plaquetas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Paxilina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Talina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Plaquetas/citologia , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Paxilina/genética , Ativação Plaquetária/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Talina/genética , Trombose/genética , Trombose/metabolismo , Trombose/patologia
3.
Cell Commun Signal ; 17(1): 101, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429758

RESUMO

BACKGROUND: Previously sharpin has been identified as an endogenous inhibitor of ß1-integrin activation by directly binding to a conserved region in the cytoplasmic tails (CTs) of the integrin ß1-associated α subunits. METHODS: Here we employed biochemical approaches and cellular analyses to evaluate the function and molecular mechanism of the sharpin-kindlin-1 complex in regulating ß1-integrin activation. RESULTS: In this study, we found that although the inhibition of sharpin on ß1-integrin activation could be confirmed, sharpin had no apparent effect on integrin αIIbß3 activation in CHO cell system. Notably, a direct interaction between sharpin and the integrin ß1 CT was detected, while the interaction of sharpin with the integrin αIIb and the ß3 CTs were substantially weaker. Importantly, sharpin was able to inhibit the talin head domain binding to the integrin ß1 CT, which can mechanistically contribute to inhibiting ß1-integrin activation. Interestingly, we also found that sharpin interacted with kindlin-1, and the interaction between sharpin and the integrin ß1 CT was significantly enhanced when kindlin-1 was present. Consistently, we observed that instead of acting as an activator, kindlin-1 actually suppressed the talin head domain mediated ß1-integrin activation, indicating that kindlin-1 may facilitate recruitment of sharpin to the integrin ß1 CT. CONCLUSION: Taken together, our findings suggest that sharpin may complex with both kindlin-1 and the integrin ß1 CT to restrict the talin head domain binding, thus inhibiting ß1-integrin activation.


Assuntos
Proteínas de Transporte/metabolismo , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células 3T3 , Animais , Células CHO , Cricetulus , Camundongos , Transdução de Sinais
4.
Blood ; 126(3): 373-7, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26056166

RESUMO

Kindlin-3 essentially supports integrin activation in blood cells. Absence of kindlin-3 in humans causes leukocyte adhesion deficiency-III characterized with severe bleeding disorder and recurrent infections. Previously, we generated kindlin-3 knock-in (K3KI) mice carrying an integrin-interaction disrupting mutation in kindlin-3 and verified the functional significance of the binding of kindlin-3 to integrin αIIbß3 in platelets. Here, using K3KI mice, we functionally evaluate the crosstalk between kindlin-3 and ß2-integrins in neutrophils. Although the kindlin-3 mutant in K3KI neutrophils is normally expressed, its binding ability to ß2-integrins in neutrophils is disabled. In vitro and in vivo analyses disclose that ß2-integrin-mediated K3KI neutrophil adhesion and recruitment are significantly suppressed. Interestingly, the ability of releasing neutrophil extracellular traps (NETs) from K3KI neutrophils is also compromised. Substantially, a peptide derived from the integrin ß2 cytoplasmic tail that can inhibit the interaction between kindlin-3 and ß2-inegrins significantly jeopardizes NET release without affecting neutrophil adhesion and recruitment under the experimental conditions. These findings suggest that crosstalk between kindlin-3 and ß2-integrins in neutrophils is required for supporting both neutrophil recruitment and NET release, but the involved regulatory mechanisms in these two cellular events might be differential, thus providing a novel therapeutic concept to treat innate immune-related diseases.


Assuntos
Plaquetas/imunologia , Antígenos CD18/metabolismo , Proteínas do Citoesqueleto/metabolismo , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Western Blotting , Antígenos CD18/imunologia , Adesão Celular , Células Cultivadas , Proteínas do Citoesqueleto/imunologia , Citometria de Fluxo , Camundongos , Neutrófilos/citologia , Neutrófilos/metabolismo
5.
Blood ; 123(6): 931-4, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24357731

RESUMO

Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive antibodies are central to the pathogenesis of heparin-induced thrombocytopenia (HIT). It is as yet unclear what triggers the initial induction of pathogenic antibodies. We identified B cells in peripheral blood of healthy adults that produce PF4/heparin-specific antibodies following in vitro stimulation with proinflammatory molecules containing deoxycytosine-deoxyguanosine (CpG). Similarly, B cells from unmanipulated wild-type mice produced PF4/heparin-specific antibodies following in vitro or in vivo CpG stimulation. Thus, both healthy humans and mice possess preexisting inactive/tolerant PF4/heparin-specific B cells. The findings suggest that breakdown of tolerance leads to PF4/heparin-specific B-cell activation and antibody production in patients developing HIT. Consistent with this concept, mice lacking protein kinase Cδ (PKCδ) that are prone to breakdown of B-cell tolerance produced anti-PF4/heparin antibodies spontaneously. Therefore, breakdown of tolerance can lead to PF4/heparin-specific antibody production, and B-cell tolerance may play an important role in HIT pathogenesis.


Assuntos
Formação de Anticorpos/imunologia , Anticoagulantes/efeitos adversos , Linfócitos B/imunologia , Heparina/efeitos adversos , Fator Plaquetário 4/metabolismo , Proteína Quinase C-delta/fisiologia , Trombocitopenia/imunologia , Adulto , Animais , Anticoagulantes/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Células Cultivadas , Heparina/metabolismo , Humanos , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator Plaquetário 4/imunologia , Prognóstico , Trombocitopenia/induzido quimicamente , Trombocitopenia/metabolismo
6.
Arterioscler Thromb Vasc Biol ; 34(9): 1961-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24969775

RESUMO

OBJECTIVE: Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbß3-mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin αIIbß3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbß3 in platelets in supporting integrin αIIbß3-mediated platelet functions. APPROACH AND RESULTS: We generated a strain of kindlin-3 knockin (K3KI) mice that express a kindlin-3 mutant that carries an integrin-interaction defective substitution. K3KI mice could survive normally and express integrin αIIbß3 on platelets similar to their wild-type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbß3 activation, suppressed platelet spreading and platelet aggregation, prolonged tail bleeding time, and absence of platelet-mediated clot retraction. In addition, whole blood drawn from K3KI mice showed resistance to in vitro thrombus formation and, as a consequence, K3KI mice were protected from in vivo arterial thrombosis. CONCLUSIONS: These observations demonstrate that the direct binding of kindlin-3 to integrin αIIbß3 is involved in supporting integrin αIIbß3 activation and integrin αIIbß3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation.


Assuntos
Plaquetas/fisiologia , Trombose das Artérias Carótidas/fisiopatologia , Proteínas do Citoesqueleto/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Substituição de Aminoácidos , Animais , Tempo de Sangramento , Plaquetas/ultraestrutura , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Forma Celular , Cloretos/toxicidade , Retração do Coágulo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Feminino , Compostos Férricos/toxicidade , Técnicas de Introdução de Genes , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismo
8.
Nat Genet ; 34(2): 220-5, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12717434

RESUMO

Mutations in LMAN1 (also called ERGIC-53) result in combined deficiency of factor V and factor VIII (F5F8D), an autosomal recessive bleeding disorder characterized by coordinate reduction of both clotting proteins. LMAN1 is a mannose-binding type 1 transmembrane protein localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC; refs. 2,3), suggesting that F5F8D could result from a defect in secretion of factor V and factor VIII (ref. 4). Correctly folded proteins destined for secretion are packaged in the ER into COPII-coated vesicles, which subsequently fuse to form the ERGIC. Secretion of certain abundant proteins suggests a default pathway requiring no export signals (bulk flow; refs. 6,7). An alternative mechanism involves selective packaging of secreted proteins with the help of specific cargo receptors. The latter model would be consistent with mutations in LMAN1 causing a selective block to export of factor V and factor VIII. But approximately 30% of individuals with F5F8D have normal levels of LMAN1, suggesting that mutations in another gene may also be associated with F5F8D. Here we show that inactivating mutations in MCFD2 cause F5F8D with a phenotype indistinguishable from that caused by mutations in LMAN1. MCFD2 is localized to the ERGIC through a direct, calcium-dependent interaction with LMAN1. These findings suggest that the MCFD2-LMAN1 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins.


Assuntos
Proteínas de Transporte/genética , Deficiência do Fator V/genética , Hemofilia A/genética , Hemorragia/genética , Lectinas de Ligação a Manose/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Transporte Biológico Ativo/genética , Retículo Endoplasmático/metabolismo , Deficiência do Fator V/metabolismo , Feminino , Complexo de Golgi/metabolismo , Células HeLa , Hemofilia A/metabolismo , Hemorragia/etiologia , Hemorragia/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas de Transporte Vesicular
9.
J Biol Chem ; 286(45): 39466-77, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-21940635

RESUMO

Rap1b is activated by platelet agonists and plays a critical role in integrin α(IIb)ß(3) inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that α(IIb)ß(3) outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y(12) or TXA(2) receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl(2), which elicits α(IIb)ß(3) outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin α(IIb)ß(3) outside-in signaling.


Assuntos
Plaquetas/metabolismo , Retração do Coágulo/fisiologia , Adesividade Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Proteínas rap de Ligação ao GTP/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Quelantes/farmacologia , Retração do Coágulo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Fibrinogênio/metabolismo , Indóis/farmacologia , Camundongos , Camundongos Mutantes , Selectina-P/genética , Selectina-P/metabolismo , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Pirimidinas/farmacologia , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas rap de Ligação ao GTP/genética
10.
Blood ; 113(1): 11-7, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18820129

RESUMO

The most significant complication of treatment in patients with hemophilia A is the development of alloantibodies that inhibit factor VIII activity. In the presence of inhibitory antibodies, replacement of the missing clotting factor by infusion of factor VIII becomes less effective. Once replacement therapy is ineffective, acute management of bleeding requires agents that bypass factor VIII activity. Long-term management consists of eradicating the inhibitor through immune tolerance. Despite success in the treatment of acute bleeding and inhibitor eradication, there remains an inability to predict or prevent inhibitor formation. Ideally, prediction and ultimately prevention will come with an improved understanding of how patient-specific and treatment-related factors work together to influence anti-factor VIII antibody production.


Assuntos
Fator VIII/administração & dosagem , Fator VIII/imunologia , Hemofilia A/tratamento farmacológico , Hemofilia A/imunologia , Tolerância Imunológica , Fator VIII/antagonistas & inibidores , Hemofilia A/epidemiologia , Humanos , Isoanticorpos/imunologia , Fatores de Risco
11.
J Thromb Haemost ; 19(12): 3126-3138, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34411430

RESUMO

BACKGROUND: Kindlin-3 is essential for supporting the bidirectional signaling of integrin αIIbß3 in platelets by bridging the crosstalk between integrin αIIbß3 and the cytoplasmic signaling adaptors. OBJECTIVE: In this study, we identified a previously unrecognized paxillin binding site in the pleckstrin homology (PH) domain of kindlin-3 and verified its functional significance. METHODS: Structure-based approaches were employed to identify the paxillin binding site in the PH domain of kindlin-3. In addition, the bidirectional signaling of integrin αIIbß3 were evaluated in both human and mouse platelets. RESULTS: In brief, we found that a ß1-ß2 loop in the PH domain of kindlin-3, an important part of the canonical membrane phospholipid binding pocket, was also involved in mediating paxillin interaction. Interestingly, the binding sites of paxillin and membrane phospholipids in the PH domain of kindlin-3 were mutually exclusive. Specific disruption of paxillin binding to the PH domain by point mutations inhibited platelet spreading on immobilized fibrinogen while having no inhibition on soluble fibrinogen binding to stimulated platelets. In addition, a membrane-permeable peptide derived from the ß1-ß2 loop in the PH domain of kindlin-3 was capable of inhibiting platelet spreading and clot retraction, but it had no effect on soluble fibrinogen binding to platelets and platelet aggregation. Treatment with this peptide significantly reduced thrombus formation in mice. CONCLUSION: Taken together, these findings suggest that interaction between paxillin and the PH domain of kindlin-3 plays an important role in supporting integrin αIIbß3 outside-in signaling in platelets, thus providing a novel antithrombotic target.


Assuntos
Plaquetas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Animais , Plaquetas/metabolismo , Retração do Coágulo , Proteínas do Citoesqueleto , Camundongos , Paxilina , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Domínios de Homologia à Plecstrina
12.
Res Sq ; 2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-34013243

RESUMO

Severe COVID-19 is associated with unprecedented thromboembolic complications. We found that hospitalized COVID-19 patients develop immunoglobulin Gs (IgGs) that recognize a complex consisting of platelet factor 4 and heparin similar to those developed in heparin-induced thrombocytopenia and thrombosis (HIT), however, independent of heparin exposure. These antibodies activate platelets in the presence of TLR9 stimuli, stimuli that are prominent in COVID-19. Strikingly, 4 out of 42 antibodies cloned from IgG1+ RBD-binding B cells could activate platelets. These antibodies possessed, in the heavy-chain complementarity-determining region 3, an RKH or Y5 motif that we recently described among platelet-activating antibodies cloned from HIT patients. RKH and Y5 motifs were prevalent among published RBD-specific antibodies, and 3 out of 6 such antibodies tested could activate platelets. Features of platelet activation by these antibodies resemble those by pathogenic HIT antibodies. B cells with an RKH or Y5 motif were robustly expanded in COVID-19 patients. Our study demonstrates that SARS-CoV-2 infection drives the development of a subset of RBD-specific antibodies that can activate platelets and have activation properties and structural features similar to those of the pathogenic HIT antibodies.

13.
Blood ; 111(9): 4627-36, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18319399

RESUMO

B-cell development is orchestrated by complex signaling networks. Rap1 is a member of the Ras superfamily of small GTP-binding proteins and has 2 isoforms, Rap1a and Rap1b. Although Rap1 has been suggested to have an important role in a variety of cellular processes, no direct evidence demonstrates a role for Rap1 in B-cell biology. In this study, we found that Rap1b was the dominant isoform of Rap1 in B cells. We discovered that Rap1b deficiency in mice barely affected early development of B cells but markedly reduced marginal zone (MZ) B cells in the spleen and mature B cells in peripheral and mucosal lymph nodes. Rap1b-deficient B cells displayed normal survival and proliferation in vivo and in vitro. However, Rap1b-deficient B cells had impaired adhesion and reduced chemotaxis in vitro, and lessened homing to lymph nodes in vivo. Furthermore, we found that Rap1b deficiency had no marked effect on LPS-, BCR-, or SDF-1-induced activation of mitogen-activated protein kinases and AKT but clearly impaired SDF-1-mediated activation of Pyk-2, a key regulator of SDF-1-mediated B-cell migration. Thus, we have discovered a critical and distinct role of Rap1b in mature B-cell trafficking and development of MZ B cells.


Assuntos
Linfócitos B/fisiologia , Quimiotaxia de Leucócito , Proteínas rap de Ligação ao GTP/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Animais , Linfócitos B/citologia , Quimiocina CXCL12/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Linfonodos/citologia , Camundongos , Proteínas rap de Ligação ao GTP/deficiência , Proteínas rap1 de Ligação ao GTP/deficiência
14.
J Immunol ; 181(5): 3373-83, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18714009

RESUMO

Rap1 is a small GTPase that belongs to Ras superfamily. This ubiquitously expressed GTPase is a key regulator of integrin functions. Rap1 exists in two isoforms: Rap1a and Rap1b. Although Rap1 has been extensively studied, its isoform-specific functions in B cells have not been elucidated. In this study, using gene knockout mice, we show that Rap1b is the dominant isoform in B cells. Lack of Rap1b significantly reduced the absolute number of B220(+)IgM(-) pro/pre-B cells and B220(+)IgM(+) immature B cells in bone marrow. In vitro culture of bone marrow-derived Rap1b(-/-) pro/pre-B cells with IL-7 showed similar proliferation levels but reduced adhesion to stromal cell line compared with wild type. Rap1b(-/-) mice displayed reduced splenic marginal zone (MZ) B cells, and increased newly forming B cells, whereas the number of follicular B cells was normal. Functionally, Rap1b(-/-) mice showed reduced T-dependent but normal T-independent humoral responses. B cells from Rap1b(-/-) mice showed reduced migration to SDF-1, CXCL13 and in vivo homing to lymph nodes. MZ B cells showed reduced sphingosine-1-phosphate-induced migration and adhesion to ICAM-1. However, absence of Rap1b did not affect splenic B cell proliferation, BCR-mediated activation of Erk1/2, p38 MAPKs, and AKT. Thus, Rap1b is crucial for early B cell development, MZ B cell homeostasis and T-dependent humoral immunity.


Assuntos
Formação de Anticorpos , Linfócitos B/citologia , Linfócitos B/imunologia , Quimiotaxia de Leucócito , Proteínas rap de Ligação ao GTP/fisiologia , Animais , Medula Óssea , Adesão Celular , Proliferação de Células , Células Cultivadas , Linfonodos , Camundongos , Camundongos Knockout , Baço , Linfócitos T/imunologia
15.
Trans Am Clin Climatol Assoc ; 121: 61-73; discussion 74-5, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20697550

RESUMO

Methods developed in the early 1970s to highly purify factor VIII (FVIII) from the plasma of large numbers of blood donors led, for the first time, to concentrates of FVIII that enabled hemophiliac to self-treat, providing independence and opening the way to safe surgery and other treatments. But, with the introduction of blood-borne viruses such as HIV-1 and hepatitis C viruses into the blood supply, these concentrates also transmitted HIV and hepatitis to a high percentage of hemophiliacs. Nevertheless, from the depths of the AIDS epidemic in hemophilia came extraordinary scientific advances that led to recombinant FVIII, the identification of HIV as the agent causing AIDS, the eventual development of effective treatments for AIDS, gene transfer approaches using lentiviruses, and treatments for hepatitis C. All of these have improved the lives of current and future hemophiliacs and have brought us to the threshold of a cure.


Assuntos
Infecções por HIV/história , Hemofilia A/história , Transfusão de Sangue/história , Clonagem Molecular , Fator VIII/genética , Fator VIII/isolamento & purificação , Fator VIII/uso terapêutico , Terapia Genética , Infecções por HIV/complicações , Infecções por HIV/transmissão , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Hemofilia A/complicações , Hemofilia A/terapia , Hepacivirus/isolamento & purificação , Hepacivirus/patogenicidade , História do Século XX , História do Século XXI , Humanos , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Reação Transfusional
16.
Blood Adv ; 4(13): 3128-3135, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32649767

RESUMO

Kindlin-3, a protein 4.1, ezrin, radixin, and moesin (FERM) domain-containing adaptor in hematopoietic cells, is essentially required for supporting the bidirectional integrin αIIbß3 signaling in platelets by binding to the integrin ß3 cytoplasmic tail. However, the structural details of kindlin-3's FERM domain remain unknown. In this study, we crystalized the kindlin-3's FERM domain protein and successfully solved its 3-dimensional structure. The structure shows that the 3 kindlin-3's FERM subdomains (F1, F2, and F3) compact together and form a cloverleaf-shaped conformation, which is stabilized by the binding interface between the F1 and F3 subdomains. Interestingly, the FERM domain of kindlin-3 exists as a monomer in both crystal and solution, which is different from its counterpart in kindlin-2 that is able to form a F2 subdomain-swapped dimer; nonetheless, dimerization is required for kindlin-3 to support integrin αIIbß3 activation, indicating that kindlin-3 may use alternative mechanisms for formation of a functional dimer in cells. To evaluate the functional importance of the cloverleaf-like FERM structure in kindlin-3, structure-based mutations were introduced into kindlin-3 to disrupt the F1/F3 interface. The results show that integrin αIIbß3 activation is significantly suppressed in platelets expressing the kindlin-3 mutant compared with those expressing wild-type kindlin-3. In addition, introduction of equivalent mutations into kindlin-1 and kindlin-2 also significantly compromises their ability to support integrin αIIbß3 activation in CHO cells. Together, our findings suggest that the cloverleaf-like FERM domain in kindlins is structurally important for supporting integrin αIIbß3 activation.


Assuntos
Plaquetas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Animais , Cricetinae , Cricetulus , Domínios FERM , Integrina beta3 , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética
17.
Biochem J ; 415(1): 145-54, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18540861

RESUMO

RGL2 [RalGDS (Ral guanine nucleotide dissociation stimulator)-like 2] is a member of the RalGDS family that we have previously isolated and characterized as a potential effector for Ras and the Ras analogue Rap1b. The protein shares 89% sequence identity with its mouse orthologue Rlf (RalGDS-like factor). In the present study we further characterized the G-protein-binding features of RGL2 and also demonstrated that RGL2 has guanine-nucleotide-exchange activity toward the small GTPase RalA. We found that RGL2/Rlf properties are well conserved between human and mouse species. Both RGL2 and Rlf have a putative PKA (protein kinase A) phosphorylation site at the C-terminal of the domain that regulates the interaction with small GTPases. We demonstrated that RGL2 is phosphorylated by PKA and phosphorylation reduces the ability of RGL2 to bind H-Ras. As RGL2 and Rlf are unique in the RalGDS family in having a PKA site in the Ras-binding domain, the results of the present study indicate that Ras may distinguish between the different RalGDS family members by their phosphorylation by PKA.


Assuntos
Proteínas rab de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Fatores de Transcrição/química , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas ras/metabolismo
18.
Aging (Albany NY) ; 11(17): 6951-6959, 2019 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-31477636

RESUMO

Platelets and myeloid cells cooperate to promote deep vein thrombosis (DVT). Here we evaluated the role of kindlin-3, a key integrin activator in these cells, in regulating stenosis-induced DVT in mice. DVT was significantly suppressed in mice that express a kindlin-3 mutant defective for integrin binding, showing that kindlin-3-mediated integrin signaling in blood cells is required for DVT. While platelet-specific deficiency of kindlin-3 in Kindlin-3fl/flPF4-Cre mice significantly suppressed DVT, deficiency of kindlin-3 specifically in myeloid cells in Kindlin-3fl/flLysM-Cre mice remarkably enhanced the early development of DVT, indicating that kindlin-3 in platelets and myeloid cells can play distinct roles in regulating DVT. Mechanistically, the levels of neutrophil extracellular traps (NETs) in plasma, a key DVT facilitator, were significantly elevated in Kindlin-3fl/flLysM-Cre mice upon the IVC stenosis; and treatment with either DNase I or PAD4 inhibitor could effectively compromise the enhancement of DVT in these mice, suggesting that kindlin-3 in neutrophils may affect DVT via restraining NET release. In addition, we found that the kindlin-3-integrin αIIbß3 signaling in platelets was required to promote NET release. Together, our studies reveal that kindlin-3 in platelets and myeloid cells can differentially regulate DVT through orchestrating NET release, thus providing further mechanistic insights into DVT.


Assuntos
Plaquetas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Armadilhas Extracelulares/metabolismo , Células Mieloides/metabolismo , Trombose Venosa/metabolismo , Animais , Camundongos Transgênicos
19.
J Clin Invest ; 115(3): 680-7, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15696195

RESUMO

Rap1b, an abundant small GTPase in platelets, becomes rapidly activated upon stimulation with agonists. Though it has been implicated to act downstream from G protein-coupled receptors (GPCRs) and upstream of integrin alpha IIbbeta3, the precise role of Rap1b in platelet function has been elusive. Here we report the generation of a murine rap1b knockout and show that Rap1b deficiency results in a bleeding defect due to defective platelet function. Aggregation of Rap1b-null platelets is reduced in response to stimulation with both GPCR-linked and GPCR-independent agonists. Underlying the defective Rap1b-null platelet function is decreased activation of integrin alphaIIbbeta3 in response to stimulation with agonists and signaling downstream from the integrin alpha IIbbeta3. In vivo, Rap1b-null mice are protected from arterial thrombosis. These data provide genetic evidence that Rap1b is involved in a common pathway of integrin activation, is required for normal hemostasis in vivo, and may be a clinically relevant antithrombotic therapy target.


Assuntos
Plaquetas/metabolismo , Hemostasia/fisiologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Transplante de Medula Óssea , Artérias Carótidas/patologia , Artérias Carótidas/fisiologia , Colágeno/metabolismo , AMP Cíclico/metabolismo , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Fibrinogênio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agregação Plaquetária/fisiologia , Antagonistas Purinérgicos , Fluxo Sanguíneo Regional , Proteínas rap de Ligação ao GTP/genética
20.
Thromb Res ; 122 Suppl 3: S1-2, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18710780

RESUMO

Clot dissolution using enzymes from the human fibrinolytic system has long been a goal of physicians and scientists. While recent focus has been on activators of the fibrinolytic system, new data support the use of plasmin itself. This supplement reviews the role of plasmin in fibrinolysis and locally administered plasmin as a safe and effective therapeutic.


Assuntos
Fibrinolíticos/uso terapêutico , Trombose/tratamento farmacológico , Humanos
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