A thrombin receptor function for platelet glycoprotein Ib–IX unmasked by cleavage of glycoprotein V

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V −/− platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib–IX–V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib–IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (Gq-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib–IX–V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib–IX via thrombin acting as a ligand, resulting in platelet activation.

H-induced platelet and crack formation in hydrogenated epitaxial Si/Si0.98 B0.02 /Si structures

An approach to transfer a high-quality Si layer for the fabrication of silicon-on-insulator wafers has been proposed based on the investigation of platelet and crack formation in hydrogenated epitaxialSi/Si0.98B0.02/Si structures grown by molecular-beam epitaxy. H-related defect formation during hydrogenation was found to be very sensitive to the thickness of the buried Si0.98B0.02 layer. For hydrogenated Si containing a 130nm thick Si0.98B0.02 layer, no platelets or cracking were observed in the B-doped region. Upon reducing the thickness of the buried Si0.98B0.02 layer to 3nm, localized continuous cracking was observed along the interface between the Si and the B-doped layers. In the latter case, the strains at the interface are believed to facilitate the (100)-oriented platelet formation and (100)-oriented crack propagation.

Stress-induced platelet formation in silicon:a molecular dynamics study

The effect of stress on vacancy cluster configurations in silicon is examined using molecular dynamics. At zero pressure, the shape and stability of the vacancy clusters agrees with previous atomistic results. When stress is applied the orientation of small planar clusters changes to reduce the strain energy. The preferred orientation for the vacancy clusters under stress agrees with the experimentally observed orientations of hydrogen platelets in the high stress regions of hydrogen implanted silicon. These results suggest a theory for hydrogen platelet formation. © 2005 The American Physical Society.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) PKA-mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Individual variation in in vitro efficacy of antiplatelet medication - aspirin and clopidogrel

Antiplatelet medication is known to decrease adverse effects in patients with atherothrombotic disease. However, despite ongoing antiplatelet medication considerable number of patients suffer from atherothrombotic events. The aims of the study were 1) to evaluate the individual variability in platelet functions and compare the usability of different methods in detecting it, 2) to assess variability in efficacy of antiplatelet medication with aspirin (acetylsalicylic acid) or the combination of aspirin and clopidogrel and 3) to investigate the main genetic and clinical variables as well as potential underlying mechanisms of variability in efficacy of antiplatelet medication. In comparisons of different platelet function tests in 19 healthy individuals PFA-100® correlated with traditional methods of measuring platelet function and was thus considered appropriate for testing individual variability in platelet activity. Efficacy of ongoing 100mg aspirin daily was studied in 101 patients with coronary artery disease (CAD). Aspirin response was measured with arachidonic acid (AA)-induced platelet aggregation, which reflects cyclo-oxygenase (COX)-1 dependent thromboxane (Tx) A2 formation, and PFA-100®, which evaluates platelet activation under high shear stress in the presence of collagen and epinephrine. Five percent of patients failed to show inhibition of AA-aggregation and 21% of patients had normal PFA-100® results despite aspirin and were thus considered non-responders to aspirin. Interestingly, the two methods of assessing aspirin efficacy, platelet aggregation and PFA-100®, detected different populations as being aspirin non-responders. It could be postulated that PFA-100® actually measures enhanced platelet function, which is not directly associated with TxA2 inhibition exerted by aspirin. Clopidogrel efficacy was assessed in 50 patients who received a 300mg loading dose of clopidogrel 2.5 h prior to percutaneous coronary intervention (PCI) and in 51 patients who were given a loading dose of 300mg combined with a five day treatment of 75mg clopidogrel daily mimicking ongoing treatment. Clopidogrel response was assessed with ADP-induced aggregations, due to its mechanism of action as an inhibitor of ADP-induced activation. When patients received only a loading dose of clopidogrel prior to PCI, 40% did not gain measurable inhibition of their ADP-induced platelet activity (inhibition of 10% or less). Prolongation of treatment so that all patients had reached a plateau of inhibition exerted by clopidogrel, decreased the incidence of non-responders to 20%. Polymorphisms of COX-1 and GP VI, as well as diabetes and female gender, were associated with decreased in vitro aspirin efficacy. Diabetes also impaired the in vitro efficacy of short-term clopidogrel. Decreased response to clopidogrel was associated with limited inhibition by ARMX, an antagonist of P2Y12-receptor, suggesting the reason for clopidogrel resistance to be receptor-dependent. Conclusions: Considerable numbers of CAD patients were non-responders either to aspirin, clopidogrel or both. In the future, platelet function tests may be helpful to individually select effective and safe antiplatelet medication for these patients.

Réduction des dommages myocardiques par le célécoxib suite à une ischémie transitoire chez le rat

Cette étude a été conçue afin d’évaluer l’effet d’un pré-traitement à long terme au célécoxib sur la taille d’infarctus suite à un infarctus du myocarde. Sachant que le célécoxib est un anti-inflammatoire et que des dommages myocardiques peuvent découler des processus inflammatoires, l’inhibition de l’inflammation devrait hypothétiquement réduire la taille d’un éventuel infarctus. Pour ce faire, un traitement au célécoxib (3 mg/kg/jour i.p.) ou au véhicule (DMSO 50% ; EtOH 15% ; eau distillée) a été administré chroniquement pendant 28 jours à des rats mâles Sprague-Dawley (n=18 par groupe) par pompes osmotiques ALZET. Après avoir été anesthésiés, les animaux ont été sujets à l’occlusion de l’artère coronaire gauche descendante, suivie d’une période de reperfusion de 24 heures. Les résultats démontrent que la taille de l’infarctus des animaux traités au célécoxib est significativement réduite comparativement à celle du groupe témoin (37,5±2,5% versus 48,0±2,6% de la zone à risque, p < 0,05). Par la suite, l’accumulation de neutrophiles indique une hausse de ces leucocytes pour la zone ischémique, sans toutefois discriminer entre les groupes traité et non-traité, qui contenaient aussi les couches sub-endocardique et sous-épicardique. Cependant, aucune différence significative est notée entre les groupes traité et témoin au niveau de l’expression de la prostaglandine E2 plasmatique et du facteur de nécrose tumorale alpha. D’un autre côté, l’apoptose, déterminée par le ratio de Bax/Bcl2 et par un essai TUNEL est significativement réduite pour la couche sub-endocardique de la zone à risque des animaux traités au célécoxib. Enfin, l’agrégation plaquettaire, induite à l’adénosine diphosphate et analysée dans le sang complet, suggère que le célécoxib diminue l’agrégation plaquettaire. Cette étude indique alors qu’un pré-traitement au célécoxib peut réduire la taille d’infarctus par un mécanisme impliquant l’apoptose.

Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A - studies in platelets, monocytes, and human endothelial cells

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide plus interferon-gamma-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-alpha-induced monocyte chemoattractant protein production in primary human endothelial cells.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Sistema purinérgico em plaquetas de ratos adultos e interações com o sistema renina-angiotensina

As plaquetas sangüíneas são fragmentos citoplasmáticos, oriundos da ruptura dos megacariócitos, cuja principal função está relacionada à manutenção da integridade vascular. Os nucleotídeos extracelulares, ATP e ADP, bem como a adenosina, têm sido implicados em um grande número de funções fisiológicas: o ADP é o principal fator recrutador de plaquetas, enquanto que o ATP é um inibidor competitivo da agregação induzida por ADP. A adenosina é uma molécula capaz de induzir vasodilatação e inibir a agregação plaquetária. Desta maneira, a manutenção da sinalização purinérgica normal tem se mostrado importante para o tratamento de doenças cardiovasculares. Os nucleosídeos di e trifosfatos circulantes podem ser hidrolisados por membros de várias famílias de ectonucleotidases de membrana e solúveis, incluindo as ecto-nucleosídeo trifosfato difosfoidrolases (E-NTPDases) e ecto-nucleotídeo pirofosfatase/fosfodiesterases (E-NPPs), que em conjunto com a ecto-5’-nucleotidase, levam à formação de adenosina. Na superfície das plaquetas, ambas enzimas, E-NTPDase e ecto-5’-nucleotidase, estão descritas. O sistema renina-angiotensina é o principal regulador da função renal e cardiovascular, desenvolvendo um papel fundamental na homeostasia da pressão arterial e do balanço eletrolítico. A angiotensina II (ANGII) induz fisiologicamente a ativação das plaquetas, possivelmente devido às suas propriedades vasoconstritoras. Os objetivos deste trabalho foram, portanto: 1) caracterizar cineticamente a enzima E-NPP em plaquetas de ratos, utilizando o substrato marcador p-Nph-5’TMP e 2) esclarecer, mesmo que em parte, os possíveis efeitos da ANGII sobre a hidrólise extracelular de nucleotídeos por plaquetas de ratos. No primeiro capítulo deste trabalho, descrevemos uma atividade enzimática em plaquetas de ratos que compartilha as principais características bioquímicas já descritas para as E-NPPs: pH ótimo alcalino; valores de KM e Vmax calculados de aproximadamente 106.22 ± 17.83 μM e 3.44 ± 0.18 nmol p-nitrophenol/min/mg, respectivamente; e dependência de cátions divalentes. Além disso, o AMP inibiu somente a hidrólise do p-Nph-5’TMP. Por outro lado, a azida de sódio, em altas concentrações, a angiotensina II e o cloreto de gadolínio alteraram apenas as hidrólises de ATP ou ADP ou de ambos. No segundo capítulo, mostramos que a ANGII foi capaz de aumentar as hidrólises de ATP, ADP e AMP em plaquetas em todas as doses testadas (5, 50, 500 e 5000 picomóis). Entretanto, nenhuma alteração foi observada com relação à hidrólise do p-Nph-5'TMP. Em adição, observamos um aumento na hidrólise de AMP e uma diminuição na hidrólise de p-Nph-5'TMP em plaquetas de ratos espontaneamente hipertensos (SHR) quando comparados a ratos Wistar normotensos. De maneira geral, esta dissertação traz a caracterização bioquímica da enzima E-NPP na superfície de plaquetas intactas de ratos como sendo parte de um complexo sistema para a hidrólise de nucleotídeos nestes fragmentos citoplasmáticos, podendo, assim, contribuir para o desenvolvimento de terapias antiplaquetárias e para o tratamento de doenças vasculares. Adicionalmente, apresentamos alguns resultados demonstrando interações entre os sistemas angiotensinérgico e adenosinérgico de plaquetas de ratos, o que poderá contribuir para o entendimento e o tratamento de doenças cardiovasculares como hipertensão e arteriosclerose.

Molecular characterization and phylogenetic analysis of JussuMP-I: A RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom

Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation. including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I. a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-1 metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases. (C) 2006 Elsevier B.V. All rights reserved.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Determinação do teste padrão para o diagnóstico de cães com doença de von willebrand

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)