RESUMEN
Factor XII (FXII) is a serine protease involved in multiple cascades, including the kallikrein-kinin system. It may play a role in diseases in which the downstream cascades are dysregulated, such as hereditary angioedema. Garadacimab (CSL312) is a first-in-class, fully human, monoclonal antibody targeting activated FXII (FXIIa). We describe how translational pharmacokinetic (PK) and pharmacodynamic (PD) modeling enabled dose selection for the phase I, first-in-human trial of garadacimab. The PK/PD data used for modeling were derived from preclinical PK/PD and safety studies. Garadacimab plasma concentrations rose with increasing dose, and clear dose-related PD effects were observed (e.g., a mechanism-based prolongation of activated partial thromboplastin time). The PK/PD profile from cynomolgus monkeys was used to generate minimal physiologically-based pharmacokinetic (mPBPK) models with target-mediated drug disposition (TMDD) for data prediction in cynomolgus monkeys. These models were later adapted for prediction of human data to establish dose selection. Based on the final mPBPK model with TMDD and assuming a weight of 70 kg for an adult human, a minimal inhibition (<10%) of FXIIa with a starting dose of 0.1 mg/kg garadacimab and a near maximal inhibition (>95%) at 10 mg/kg garadacimab were predicted. The phase I study is complete, and data on exposure profiles and inhibition of FXIIa-mediated kallikrein activity observed in the trial support and validate these simulations. This emphasizes the utility and relevance of translational modeling and simulation in drug development.
Asunto(s)
Angioedemas Hereditarios , Factor XIIa , Animales , Anticuerpos Monoclonales/farmacocinética , Simulación por Computador , Humanos , Macaca fascicularisRESUMEN
BACKGROUND: Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. OBJECTIVE: The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. METHODS: A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. RESULTS: These hF12KI mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12KI mice in an arterial thrombosis model without affecting bleeding times. CONCLUSION: These data establish the newly generated hF12KI mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.
Asunto(s)
Factor XII , Trombosis , Animales , Coagulación Sanguínea , Modelos Animales de Enfermedad , Factor XII/genética , Hemostasis , Ratones , Trombosis/tratamiento farmacológico , Trombosis/genéticaRESUMEN
BACKGROUND: Hemophilia B is caused by coagulation factor IX (FIX) deficiency. Recombinant fusion protein linking coagulation FIX with recombinant albumin (rIX-FP; Idelvion® ) is used for replacement therapy with an extended half-life. A previous quantitative whole-body autoradiography (QWBA) study investigating the biodistribution of rIX-FP indicated equal biodistribution, but more prolonged tissue retention compared with a marketed recombinant FIX product. OBJECTIVES: To complete and confirm the QWBA study data by directly measuring rIX-FP protein and activity levels in tissues following intravenous (i.v.) administration to normal rats and FIX-deficient (hemophilia B) mice. METHODS: After i.v. administration of rIX-FP at a dose of 2000 IU/kg, animals were euthanized at specific time points up to 72 hours postdosing. Subsequently, plasma and various tissues, which were selected based on the previous QWBA results, were harvested and analyzed for FIX antigen levels using an ELISA (both species) or an immunohistochemistry method (mice only), as well as for FIX activity levels (mice only) using a chromogenic assay. RESULTS: In rats, rIX-FP distributed extravascularly into all tissues analyzed (ie, liver, kidney, skin and knee) with peak antigen levels reached between 1 and 7 hours postdosing. In hemophilia B mice, rIX-FP tissue distribution was comparable to rats. FIX antigen levels correlated well with FIX activity readouts. CONCLUSIONS: Our results confirm QWBA data showing that rIX-FP distributes into relevant target tissues. Importantly, it was demonstrated that rIX-FP available in tissues retains its functional activity and can thus facilitate its therapeutic activity at sites of potential injury.
Asunto(s)
Hemofilia B , Roedores , Administración Intravenosa , Animales , Factor IX/metabolismo , Semivida , Hemofilia B/tratamiento farmacológico , Ratones , Ratas , Proteínas Recombinantes de Fusión/uso terapéutico , Roedores/metabolismo , Distribución TisularRESUMEN
Haemostasis including blood coagulation is initiated upon vessel wall injury and indispensable to limit excessive blood loss. However, unregulated pathological coagulation may lead to vessel occlusion, causing thrombotic disorders, most notably myocardial infarction and stroke. Furthermore, blood exposure to foreign surfaces activates the intrinsic pathway of coagulation. Hence, various clinical scenarios, such as extracorporeal membrane oxygenation, require robust anticoagulation consequently leading to an increased bleeding risk. This study aimed to further assess the antithrombotic efficacy of the activated factor XII (FXIIa) inhibitor, rHA-Infestin-4, in several thrombosis models. In mice, rHA-Infestin-4 decreased occlusion rates in the mechanically-induced arterial (Folt's) and the FeCl3 -induced venous thrombosis model. rHA-Infestin-4 also protected from FeCl3 -induced arterial thrombosis and from stasis-prompted venous thrombosis in rabbits. Furthermore, rHA-Infestin-4 prevented occlusion in the arterio-venous shunt model in mice and rabbits where thrombosis was induced via a foreign surface. In contrast to heparin, the haemostatic capacity in rabbits was unaffected by rHA-Infestin-4. Using rodent and non-rodent species, our data demonstrate that the FXIIa inhibitor rHA-Infestin-4 decreased arterial, venous and foreign surface-induced thrombosis without affecting physiological haemostasis. Hence, we provide further evidence that targeting FXIIa represents a potent yet safe antithrombotic treatment approach, especially in foreign surface-triggered thrombosis.
Asunto(s)
Factor XIIa/antagonistas & inhibidores , Proteínas de Insectos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Albúmina Sérica/farmacología , Trombosis/tratamiento farmacológico , Animales , Arteriopatías Oclusivas/tratamiento farmacológico , Arteriopatías Oclusivas/etiología , Modelos Animales de Enfermedad , Fibrinolíticos/farmacología , Hemostasis/efectos de los fármacos , Proteínas de Insectos/uso terapéutico , Cinética , Ratones , Conejos , Proteínas Recombinantes de Fusión/uso terapéutico , Albúmina Sérica/uso terapéutico , Albúmina Sérica Humana , Trombosis/etiología , Resultado del Tratamiento , Trombosis de la Vena/tratamiento farmacológico , Trombosis de la Vena/etiologíaRESUMEN
Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)ß3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)ß3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)ß3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)ß3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)ß3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)ß3.
Asunto(s)
Plaquetas/metabolismo , Factor XIII/metabolismo , Fibrina/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombastenia/sangre , Animales , Coagulación Sanguínea , Plaquetas/efectos de los fármacos , Plaquetas/patología , Venenos de Crotálidos/farmacología , Factor Va/química , Factor Va/metabolismo , Factor XIII/química , Factor Xa/química , Factor Xa/metabolismo , Fibrina/química , Fibrinógeno/química , Fibrinógeno/metabolismo , Humanos , Lectinas Tipo C , Ratones , Ratones Noqueados , Sondas Moleculares/química , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Cultivo Primario de Células , Unión Proteica , Trombastenia/patología , Trombina/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. METHODS: For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. RESULTS: Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. CONCLUSIONS: With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.
Asunto(s)
Factor XIIa/antagonistas & inhibidores , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Proteínas de Insectos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Daño por Reperfusión/prevención & control , Inhibidores de Serina Proteinasa/farmacología , Albúmina Sérica/farmacología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/patología , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Factor XIIa/metabolismo , Proteínas de Insectos/uso terapéutico , Masculino , Ratas , Proteínas Recombinantes de Fusión/uso terapéutico , Prueba de Desempeño de Rotación con Aceleración Constante , Inhibidores de Serina Proteinasa/uso terapéutico , Albúmina Sérica/uso terapéutico , Albúmina Sérica HumanaRESUMEN
Human plasma-derived C1-esterase inhibitor (C1-INH) is an efficacious and safe treatment for hereditary angioedema. However, thrombotic events in subjects treated with C1-INH at recommended or off-label, high doses have been reported. In this study, we addressed the potential prothrombotic risk of C1-INH treatment in high doses using a non-clinical rabbit model. Following intravenous infusion of C1-INH to rabbits at doses up to 800 IU/kg, the exposure and the pharmacodynamic efficacy of C1-INH in rabbits were confirmed by activity measurements of C1-esterase, and coagulation factors XIa and XIIa, respectively. Potential prothrombotic effects were assessed following induction of venous and arterial thrombosis using in vivo models of venous and arterial stasis, complemented by various in vitro assays of coagulation markers. Administration of C1-INH at doses up to 800 IU/kg did not potentiate thrombus formation during venous stasis. In contrast, inhibition of arterial occlusion was observed upon C1-INH administration when compared with isotonic saline treatment, indicating antithrombotic rather than prothrombotic activity of high dose C1-INH treatment in vivo. This was further confirmed in vitro by decreased thrombin generation, increased activated partial thromboplastin time, clotting time and clot formation time, and inhibition of platelet aggregation. No relevant changes in fibrinolysis or in the levels of thrombin-antithrombin complexes, and prothrombin fragment 1+2 were observed upon high dose C1-INH treatment. The data suggest that treatment of healthy rabbits with high doses of C1-INH could potentially inhibit coagulation and thrombus formation rather than induce a prothrombotic risk.
Asunto(s)
Arteriopatías Oclusivas/inducido químicamente , Proteína Inhibidora del Complemento C1/toxicidad , Trombosis de la Vena/inducido químicamente , Animales , Pruebas de Coagulación Sanguínea , Proteína Inhibidora del Complemento C1/administración & dosificación , Proteína Inhibidora del Complemento C1/farmacocinética , Proteína Inhibidora del Complemento C1/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Factor XIIa/análisis , Factor XIa/análisis , Arteria Femoral , Fibrinólisis/efectos de los fármacos , Humanos , Infusiones Intravenosas , Sistema Calicreína-Quinina/efectos de los fármacos , Sistema Calicreína-Quinina/fisiología , Agregación Plaquetaria/efectos de los fármacos , Conejos , Tromboelastografía , Trombina/biosíntesisRESUMEN
OBJECTIVE: Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor-factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo. APPROACH AND RESULTS: Plaque rupture after ultrasound treatment evoked immediate formation of subocclusive thrombi in the carotid arteries of Apoe(-/-) mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas. CONCLUSIONS: The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor-FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stability.
Asunto(s)
Enfermedades de la Aorta/complicaciones , Aterosclerosis/complicaciones , Coagulación Sanguínea , Plaquetas/metabolismo , Enfermedades de las Arterias Carótidas/complicaciones , Factor XII/metabolismo , Placa Aterosclerótica , Trombosis/etiología , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/patología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Colesterol en la Dieta , Modelos Animales de Enfermedad , Factor VIIa/metabolismo , Factor XI/metabolismo , Factor XII/genética , Deficiencia del Factor XII/sangre , Deficiencia del Factor XII/genética , Factor XIIa/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Rotura Espontánea , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/genética , Trombosis/patología , Factores de TiempoRESUMEN
Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.
Asunto(s)
Endotelio Linfático/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Endotelio Linfático/inmunología , Femenino , Regulación de la Expresión Génica , Uniones Intercelulares/genética , Uniones Intercelulares/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Lisofosfolípidos/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
OBJECTIVE: Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif-bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. APPROACH AND RESULTS: We investigated whether both (hem)immunoreceptor tyrosine activation motif-bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2-depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. CONCLUSIONS: These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti-CLEC-2-based agents in the prevention of thrombotic diseases.
Asunto(s)
Hemostasis , Lectinas Tipo C/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Trombosis/prevención & control , Animales , Lectinas Tipo C/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidoresRESUMEN
Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte- and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoA-deficient platelets in response to G(13)-coupled agonists was abolished, and it was impaired in response to G(q) stimulation. Similarly, RhoA was required for efficient secretion of α and dense granules downstream of G(13) and G(q). Furthermore, RhoA was essential for integrin-mediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis.
Asunto(s)
Plaquetas/patología , Hemostasis , Megacariocitos/metabolismo , Activación Plaquetaria , Trombocitopenia/fisiopatología , Trombosis/prevención & control , Proteínas de Unión al GTP rho/metabolismo , Animales , Tiempo de Sangría , Plaquetas/efectos de los fármacos , Infarto Encefálico/prevención & control , Señalización del Calcio , Forma de la Célula , Tamaño de la Célula , Retracción del Coagulo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Hemostasis/efectos de los fármacos , Cinética , Megacariocitos/efectos de los fármacos , Ratones , Ratones Noqueados , Activación Plaquetaria/efectos de los fármacos , Recuento de Plaquetas , Trombocitopenia/sangre , Trombocitopenia/metabolismo , Trombocitopenia/patología , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoARESUMEN
Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti-CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2-deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.
Asunto(s)
Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Adhesividad Plaquetaria , Agregación Plaquetaria , Trombosis/metabolismo , Animales , Anticuerpos/farmacología , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Lectinas Tipo C/antagonistas & inhibidores , RatonesRESUMEN
Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.