RESUMO
The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Venenos de Crotalídeos/uso terapêutico , Fibrinolíticos/uso terapêutico , Lectinas Tipo C/uso terapêutico , Complexo Glicoproteico GPIb-IX de Plaquetas/antagonistas & inibidores , Venenos de Serpentes/uso terapêutico , Animais , Coagulação Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacocinética , Crotalinae , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacocinética , Voluntários Saudáveis , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/isolamento & purificação , Modelos Moleculares , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Ligação Proteica , Conformação Proteica , Ristocetina/farmacologia , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Venenos de Serpentes/farmacocinética , Relação Estrutura-Atividade , Trombina/farmacologia , Trombose/prevenção & controle , Fator de von Willebrand/química , Fator de von Willebrand/metabolismoRESUMO
Cell adhesion plays a pivotal role in various biological processes, e.g., immune responses, cancer metastasis, and stem cell differentiation. The adhesion behaviors depend subtly on the binding kinetics of receptors and ligands restricted at the cell-substrate interfaces. Although much effort has been directed toward investigating the kinetics of adhesion molecules, the role of the glycocalyx, anchored on cell surfaces as an exterior layer, is still unclear. In this paper, we propose a theoretical approach to study the collective binding kinetics of a few and a large number of binders in the presence of the glycocalyx, representing the cases of initial and mature adhesions of cells, respectively. The analytical results are validated by finding good agreement with our Monte Carlo simulations. In the force loading case, the on-rate and affinity increase as more bonds form, whereas this cooperative effect is not observed in the displacement loading case. The increased thickness and stiffness of the glycocalyx tend to decrease the affinity for a few bonds, while they have less influence on the affinity for a large number of bonds. Moreover, for a flexible membrane with thermally-excited shape fluctuations, the glycocalyx is exhibited to promote the formation of bond clusters, mainly due to the cooperative binding of binders. This study helps to understand the cooperative kinetics of adhesion receptors under physiologically relevant loading conditions and sheds light on the novel role of the glycocalyx in cell adhesion.
Assuntos
Glicocálix/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Agregados Proteicos , Elasticidade , Cinética , Método de Monte Carlo , Ligação Proteica , TermodinâmicaRESUMO
BACKGROUND: Acquired and inherited bleeding disorders may present in the neonatal period with devastating lifelong effects. Diagnosing bleeding disorders in the neonatal population could aid in preventing and treating the associated complications. However, currently available platelet function testing is limited in neonates, owing to difficulties in obtaining an adequate blood volume, a lack of normal reference ranges, and an incomplete understanding of the neonatal platelet functional phenotype. OBJECTIVE: To develop small-volume, whole blood platelet function assays in order to quantify and compare neonatal and adult platelet function. METHODS AND RESULTS: Peripheral blood was obtained from healthy, full-term neonates at 24 h of life. Platelet activation, secretion and aggregation were measured via flow cytometry. Platelet adhesion and aggregation were assessed under static and flow conditions. As compared with adult platelets, peripheral neonatal platelet P-selectin expression and integrin glycoprotein IIbIIIa activation were significantly reduced in response to the G-protein-coupled receptor (GPCR) agonists thrombin receptor activator peptide-6 (TRAP-6), ADP, and U46619, and the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway agonists collagen-related peptide (CRP) and rhodocytin. Neonatal platelet aggregation was markedly reduced in response to TRAP-6, ADP, U46619, CRP and rhodocytin as compared with adult platelets. The extents of neonatal and adult platelet adhesion and aggregate formation under static and shear conditions on collagen and von Willebrand factor were similar. CONCLUSIONS: As compared with adult platelets, we found that neonatal platelet activation and secretion were blunted in response to GPCR or ITAM agonists, whereas the extent of neonatal platelet adhesion and aggregate formation was similar to that of adult platelets.
Assuntos
Plaquetas/citologia , Ativação Plaquetária , Adesividade Plaquetária , Agregação Plaquetária , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/química , Difosfato de Adenosina/química , Adulto , Proteína C-Reativa/química , Separação Celular , Citometria de Fluxo , Glicoproteínas/química , Hemorragia/sangue , Humanos , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Recém-Nascido , Lectinas Tipo C/química , Oligopeptídeos/química , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Receptores Acoplados a Proteínas G/agonistas , Transdução de SinaisRESUMO
MOTIVATION: Abrupt reduction/resumption of thermal fluctuations of a force probe has been used to identify association/dissociation events of protein-ligand bonds. We show that off-rate of molecular dissociation can be estimated by the analysis of the bond lifetime, while the on-rate of molecular association can be estimated by the analysis of the waiting time between two neighboring bond events. However, the analysis relies heavily on subjective judgments and is time-consuming. To automate the process of mapping out bond events from thermal fluctuation data, we develop a hidden Markov model (HMM)-based method. RESULTS: The HMM method represents the bond state by a hidden variable with two values: bound and unbound. The bond association/dissociation is visualized and pinpointed. We apply the method to analyze a key receptor-ligand interaction in the early stage of hemostasis and thrombosis: the von Willebrand factor (VWF) binding to platelet glycoprotein Ibα (GPIbα). The numbers of bond lifetime and waiting time events estimated by the HMM are much more than those estimated by a descriptive statistical method from the same set of raw data. The kinetic parameters estimated by the HMM are in excellent agreement with those by a descriptive statistical analysis, but have much smaller errors for both wild-type and two mutant VWF-A1 domains. Thus, the computerized analysis allows us to speed up the analysis and improve the quality of estimates of receptor-ligand binding kinetics.
Assuntos
Algoritmos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Cinética , Ligantes , Cadeias de Markov , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Ligação Proteica , Temperatura , Fator de von Willebrand/químicaRESUMO
The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.