In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

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.

Assessment of neonatal platelet adhesion, activation, and aggregation.

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.

Individual venom profiling of Crotalus durissus terrificus specimens from a geographically limited region: crotamine assessment and captivity evaluation on the biological activities.

Crotalus durissus terrificus (Cdt) venom major components comprise crotoxin, crotamine, gyroxin and convulxin. Crotamine exerts a myotoxic action, among others, but its expression varies even amid snakes from the same region. Biochemical, enzymatic and pharmacological variations of venoms may be associated with the geography, climate, gender, age, and diet, as well as captivity time and venom extraction intervals. The present study aimed to characterize the Cdt venom from the Botucatu region, (SP, Brazil), by assessing its biochemical, pharmacological and enzymatic properties. Venoms from newly captured snakes and already-captured animals were characterized comparatively to verify the sexual, environmental (length of captivity) and ontogenetic variations that could influence the venom composition. Protein concentration, SDS-PAGE and RP-HPLC were performed and the coagulant, toxic (LD50) and crotamine activities were assayed. Individual SDS-PAGE analyses (315 samples) were performed and the biological activities of the venom of 60 adults (captive and newly captured males and females) and 18 newborns were compared with the Brazilian Reference Venom. Crotamine was found in 39.7% (125/315) of the samples, as determined by SDS-PAGE and RP-HPLC. Protein concentration differed significantly between adults (75%) and newborns (60%). RP-HPLC and SDS-PAGE analyses showed highly variable protein concentration and copious crotoxin isoforms; however, the LD50 values decreased during the captivity time. Cdt venom biological activities were similar among adult groups, but diminished during the captivity period. The current findings demonstrate that venoms vary significantly in terms activity and protein concentration, despite originating from the same specie and region.

Assignment of disulfide-linked peptides using automatic a1 ion recognition.

We present a novel approach for the assignment of peptides containing disulfide linkages. Dimethyl labeling is introduced to generate labeled peptides which exhibit enhanced a1 ion signals during MS/MS fragmentation. For disulfide-linked peptides, multiple a1 ions can be observed due to multiple N-termini. This distinct feature allows sieving out the disulfide-linked peptides; meanwhile, the N-terminal amino acids can be identified. With such information, the number of possible peptide combinations involved in a disulfide bond dramatically narrows down. Furthermore, we developed a computational algorithm to perform target a1 ion screening followed by molecular weight matching of cysteine-containing peptides with specific amino acids at the N-termini. Once the protein sequence and the peak list from a LC-MS/MS survey scan of labeled peptides are imported, the identities of disulfide-linked peptides can be readily obtained. The presented approach is simple and straightforward, offering a valuable tool for protein structural characterization.

Force-mediated dissociation of proteoglycan aggregate in articular cartilage.

Proteoglycan aggregate is the primary component in articular cartilage responsible for resisting compressive loading. It consists of a core molecule of hyaluronan and a number of side chains of aggrecan bound to hyaluronan non-covalently. The loss of aggrecan from articular cartilage is considered to be a major factor in the development of osteoarthritis. Though enzymatic digestion of aggrecan is believed to be responsible for the release of aggrecan from osteoarthritic cartilage, other mechanisms, such as direct force-mediated detachment of aggrecan from hyaluronan may also be involved. In this study, the rupture force of the single bond between hyaluronan and aggrecan in articular cartilage was directly quantified using experimental measurement and Monte Carlo simulation. Low rupture force of this bond, as determined in this study suggested a possible direct force-mediated detachment of aggrecan from proteoglycan aggregate in osteoarthritic cartilage.