RESUMEN
Cellular and humoral immunity are both required for SARS-CoV-2 infection recovery and vaccine efficacy. The factors affecting mRNA vaccination-induced immune responses, in healthy and fragile subjects, are still under investigation. Thus, we monitored the vaccine-induced cellular and humoral immunity in healthy subjects and cancer patients after vaccination to define whether a different antibody titer reflected similar rates of cellular immune responses and if cancer has an impact on vaccination efficacy. We found that higher titers of antibodies were associated with a higher probability of positive cellular immunity and that this greater immune response was correlated with an increased number of vaccination side effects. Moreover, active T-cell immunity after vaccination was associated with reduced antibody decay. The vaccine-induced cellular immunity appeared more likely in healthy subjects rather than in cancer patients. Lastly, after boosting, we observed a cellular immune conversion in 20% of subjects, and a strong correlation between pre- and post-boosting IFN-γ levels, while antibody levels did not display a similar association. Finally, our data suggested that integrating humoral and cellular immune responses could allow the identification of SARS-CoV-2 vaccine responders and that T-cell responses seem more stable over time compared to antibodies, especially in cancer patients.
Asunto(s)
COVID-19 , Inmunidad Humoral , Humanos , Vacunas contra la COVID-19 , SARS-CoV-2 , COVID-19/prevención & control , Vacunación , Anticuerpos , Inmunidad Celular , Anticuerpos AntiviralesRESUMEN
Type 2N is a rare von Willebrand disease (VWD) variant involving an impairment in the factor VIII (FVIII) carrier function of von Willebrand factor (VWF). It has a phenotype that mimics hemophilia A, and FVIII binding to VWF (VWF:FVIIIB) is tested to differentiate between the two disorders. Type 2N VWF defects may also be associated with quantitative VWF mutations (type 2N/type 1), further complicating the identification of cases. We report on a new quantitative VWF mutation (c.2547-1G > T) revealed by a p.R854Q type 2N mutation acting as homozygous despite being carried as a heterozygous defect. The proband had near-normal VWF levels (initially ruling out a defective VWF synthesis) and slightly reduced FVIII levels, while a VWF:FVIIIB test showed significantly reduced binding. Routine tests on type 2N homozygotes or heterozygotes combined with quantitative VWF defects in our cohort showed reduced FVIII levels in both groups, but it was only in the former that the FVIII/VWF antigen (VWF:Ag) ratio was always significantly reduced. The two tests are therefore not enough to identify all forms of type 2N VWD. While relatives of type 2N homozygotes usually have normal FVIII levels and FVIII/VWF:Ag ratios, relatives of type 2N/type 1 may have high FVIII/VWF:Ag ratios, but their VWF:FVIIIB and/or VWF:FVIIIB/VWF:Ag ratios are always low. Measuring FVIII and VWF levels may therefore suggest type 2N VWD in patients carrying type 2N mutations alone, but not in type 2N combined with quantitative VWF defects. The VWF:FVIIIB test should consequently be included when exploring VWF function, whatever VWD patient's phenotype.
Asunto(s)
Hemofilia A , Enfermedad de von Willebrand Tipo 2 , Enfermedades de von Willebrand , Factor VIII , Humanos , Mutación , Factor de von WillebrandRESUMEN
BACKGROUND AND AIMS: Monitoring the immune response against SARS-CoV-2 is pivotal in the evaluation of long-term vaccine efficacy. Immunoglobulin G (IgG) antibodies represent an advisable tool to reach this goal, especially for the still poorly defined antibody trend induced by the new class of mRNA vaccines against SARS-CoV-2. MATERIALS AND METHODS: Anti-Spike RBD IgG antibodies were monitored in a cohort of healthcare workers at CRO Aviano, National Cancer Institute, through MAGLUMI® chemiluminescence assay, at 1 and 4 months after full-schedule of BNT162b2 or mRNA-1273 vaccination. RESULTS: At 1 month after vaccination, 99.9% of 767 healthcare workers showed a reactive antibody response, which was inversely correlated with age, and positively associated with a previous history of COVID-19, and mRNA-1273 vaccination. Serological response was maintained in 99.6% of the 516 subjects monitored also at follow-up. An antibody decay from 559.8 AU/mL (IQR 359.7-845.7) to 92.7 AU/mL (IQR 65.1-148.6; p < 0.001) was observed, independently from age and sex. CONCLUSION: Our data supported the ability of SARS-CoV-2 mRNA vaccines to induce at least a 4 months-lasting IgG response, even outside the rules of clinical trials. The antibody decay observed at follow-up suggested to deepen the immune response characterization to identify subjects with low anti-SARS-CoV-2 immunity possibly requiring a vaccination boost.
Asunto(s)
COVID-19 , SARS-CoV-2 , Vacuna nCoV-2019 mRNA-1273 , Vacuna BNT162 , Vacunas contra la COVID-19 , Personal de Salud , Humanos , Inmunoglobulina G , Vacunación , Eficacia de las Vacunas , Vacunas de ARNmRESUMEN
BACKGROUND: Local excision (LE) is currently one of the most effective methods used in cases of large benign polyps, not suitable for endoscopic treatment, or early-stage neoplasms. LE is also alternative to anterior rectal resection in selected patients suffering from major comorbidities and limits for major abdominal procedure. Furthermore, LE results in less pain, reduced impact on bowel function, shorter duration of hospital stay, and lower rates of morbidity, mortality and stoma creation. In particular, early data on transanal minimally invasive surgery (TAMIS) are promising, but they come from single centre case series related to small groups of patients and more data are needed to draw a final conclusion on the safety of this novel approach for transanal resection. CASE PRESENTATION: A 62-year-old woman, following a positive faecal occult blood test and with unremarkable medical history, was admitted to hospital for excision of a large flat neoplastic lesion. Endoscopic biopsy demonstrated a tubular adenoma with high-grade dysplasia and was decided to proceed with surgical excision by TAMIS. After surgery, short-term outcomes revealed prolonged activated partial thromboplastin time, undetectable factor XII activity, fever, and partial dehiscence of rectal wall defect suture. Cross-mixing studies of patient plasma show no correction in either the immediate or incubated activated partial thromboplastin time, indicating the presence of an acquired factor XII inhibitor. Activated partial thromboplastin time and factor XII improved in the following weeks without any specific therapy in addition to antibiotic therapy. CONCLUSION: This is the first report in which acquired inhibitor of coagulation factor XII is associated with a specific surgical procedure. This case has shown how trans-anal excision of rectal lesions, even when performed by minimally invasive means such as in case of TAMIS, is not free of complications. We consider the acute infection, resulting from early dehiscence of the suture, the trigger in an abnormal immune response, and inhibitor development.
Asunto(s)
Pólipos Adenomatosos/cirugía , Deficiencia del Factor XII/etiología , Neoplasias del Recto/cirugía , Dehiscencia de la Herida Operatoria/etiología , Cirugía Endoscópica Transanal/efectos adversos , Pólipos Adenomatosos/patología , Canal Anal/cirugía , Traslocación Bacteriana , Factor XII/análisis , Deficiencia del Factor XII/sangre , Deficiencia del Factor XII/diagnóstico , Femenino , Heparina de Bajo-Peso-Molecular , Humanos , Persona de Mediana Edad , Procedimientos Quirúrgicos Mínimamente Invasivos/efectos adversos , Tiempo de Tromboplastina Parcial , Pronóstico , Neoplasias del Recto/patologíaRESUMEN
Elevated plasma fibrinogen levels and tumor progression in patients with gastric cancer (GC) have been largely reported. However, distinct fibrinogen chains and domains have different effects on coagulation, inflammation, and angiogenesis. The aim of this study was to characterize fibrinogen ß chain (FGB) in GC tissues. Retrospectively we analyzed the data of matched pairs of normal (N) and malignant tissues (T) of 28 consecutive patients with GC at diagnosis by combining one- and two-dimensional electrophoresis (1DE and 2DE) with immunoblotting and mass spectrometry together with two-dimensional difference in gel electrophoresis (2D-DIGE). 1DE showed bands of the intact FGB at 50 kDa and the cleaved forms containing the fragment D at ~37-40 kDa, which corresponded to 19 spots in 2DE. In particular, spot 402 at ~50 kDa and spots 526 and 548 at ~37 kDa were of interest by showing an increased expression in tumor tissues. A higher content of spot 402 was associated with stomach antrum, while spots 526 and 548 amounts correlated with corpus and high platelet count (>208 × 108/L). The quantification of FGB and cleaved products may help to further characterize the interconnections between GC and platelet/coagulation pathways.
Asunto(s)
Carcinoma/metabolismo , Proteínas Portadoras/metabolismo , Fibrinógeno/metabolismo , Subunidades de Proteína/metabolismo , Proteómica , Neoplasias Gástricas/metabolismo , Adulto , Anciano , Biología Computacional/métodos , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Femenino , Fibrinógeno/química , Humanos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana Edad , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Reproducibilidad de los ResultadosRESUMEN
Nitric oxide (NO) exerts vasodilatatory, antiplatelet, antioxidant, and antiproliferative effects. Endothelium-derived NO has been shown to be of crucial importance in cardiovascular protection, whereas evidence that NO is synthesized by platelets and regulates platelet function is still controversial. By using a sensitive and specific fluorescent probe, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), we visualized NO production in individual platelets undergoing adhesion on a collagen substrate under flow conditions. NO production, monitored in real time, was dependent on the shear rates applied, increasing with the raising of the shear rates. Furthermore, NO production increased in the presence of l-arginine (nitric-oxide synthase [NOS] substrate), and it decreased in the presence of L-NG-monomethyl arginine (L-NMMA) (NOS inhibitor) but not of D-NG-monomethyl arginine (D-NMMA) (L-NMMA-inactive enantiomer). Platelet deposition, measured with mepacrine-labeled platelets, was inversely related to NO production. A correlation was evident between Ca(++) elevation and NO production, suggesting that platelet NO formation is triggered by intracytoplasmic Ca(++) elevation. Simultaneous measurement of NO and Ca(++) indicated that NO production in individual platelets is preceded by Ca(++) elevations, with a lag phase of 33 ± 9.5 s. Our studies provide the first direct demonstration of platelet NO production triggered by the interaction with an activating surface under flow and suggest that intraplatelet Ca(++) elevation elicits the production of NO which, in turn, modulates thrombus size.
Asunto(s)
Plaquetas/metabolismo , Óxido Nítrico/metabolismo , Adhesividad Plaquetaria/fisiología , Animales , Velocidad del Flujo Sanguíneo , Plaquetas/citología , Calcio/metabolismo , Colágeno/farmacología , Inhibidores Enzimáticos/farmacología , Fluoresceínas/farmacocinética , Masculino , Ratones , Ratones Noqueados , Adhesividad Plaquetaria/efectos de los fármacos , Quinacrina/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
OBJECTIVE: Platelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease. METHODS AND RESULTS: We therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen. CONCLUSIONS: Our data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.
Asunto(s)
Plaquetas/enzimología , Infarto del Miocardio/enzimología , Proteína Quinasa C-epsilon/sangre , Anciano , Secuencia de Bases , Estudios de Casos y Controles , Cartilla de ADN , ADN Complementario , Femenino , Citometría de Flujo , Humanos , Masculino , Infarto del Miocardio/sangre , Activación Plaquetaria , Proteína Quinasa C-epsilon/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We studied how integrin alpha2beta1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca(2+) concentration ([Ca(2+)](i)) of FLUO 3-AM-labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca(2+)](i) oscillations. Rapid alpha-like peaks were unaffected by the membrane-impermeable Ca(2+) chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting gamma-like peaks were always preceded by at least one alpha-like peak and abolished by intracellular or extracellular Ca(2+) chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca(2+) responses. Human or mouse platelets lacking GPVI function exhibited alpha-like but not gamma-like Ca(2+) peaks, whereas those lacking alpha2beta1 showed markedly reduced to absent alpha-like and no gamma-like Ca(2+) peaks. Specific alpha2beta1 ligation induced alpha-like but not gamma-like peaks. Thus, alpha2beta1 may generate Ca(2+) signals that are reinforced by GPVI and required for subsequent longer-lasting Ca(2+) oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of alpha2beta1 in response to collagen stimulation.
Asunto(s)
Circulación Sanguínea/fisiología , Plaquetas/metabolismo , Señalización del Calcio/fisiología , Integrina alfa2beta1/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Animales , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Células Cultivadas , Cromonas/farmacología , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Integrina alfa2beta1/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adhesividad Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/genética , Adhesividad Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/genética , Factores de TiempoRESUMEN
A versatile and automated image processing technique and data extraction procedure from videomicroscopic data is presented. The motivation is a detailed quantification of blood platelet adhesion from laminar flow onto a surface. The characteristics of the system under observation (type of cells, their speed of movement, and the quality of the optical image to analyze) provided the criteria for developing a new procedure enabling tracking for long image sequences. Specific features of the novel method include: automatic segmentation methodology which removes operator bias; platelet recognition across the series of images based on a probability density function (two-dimensional, Gaussian-like) tailored to the physics of platelet motion on the surface; options to automatically tune the procedure parameters to explore different applications; integrated analysis of the results (platelet trajectories) to obtain relevant information, such as deposition and removal rates, displacement distributions, pause times and rolling velocities. Synthetic images, providing known reference conditions, are used to test the method. The algorithm operation is illustrated by application to images obtained by fluorescence microscopy of the interaction between platelets and von Willebrand factor-coated surfaces in parallel-plate flow chambers. Potentials and limits are discussed, together with evaluation of errors resulting from an inaccurate tracking.
Asunto(s)
Algoritmos , Plaquetas/citología , Plaquetas/fisiología , Citometría de Flujo/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Fluorescente/métodos , Microscopía por Video/métodos , Adhesividad Plaquetaria/fisiología , Animales , Velocidad del Flujo Sanguíneo/fisiología , Técnicas de Cultivo de Célula/métodos , Movimiento Celular/fisiología , Células Cultivadas , HumanosRESUMEN
We have investigated the role of adenosine diphosphate (ADP) receptors in the adhesion, activation, and aggregation of platelets perfused over immobilized von Willebrand factor (VWF) under high shear stress. Blocking P2Y(1) prevented stable platelet adhesion and aggregation, indicative of a complete inhibition of alpha(IIb)beta(3) activation, and decreased the duration of transient arrests from 5.9 seconds +/- 2.8 seconds to 1.2 seconds +/- 0.8 seconds; in contrast, blocking P2Y(12) inhibited only the formation of larger aggregates. Moreover, blocking P2Y(1) decreased the proportion of platelets showing early intracytoplasmic Ca(++) elevations (alpha/beta peaks) from 20.6% +/- 1.6% to 14.6% +/- 1.5% (P < .01), and the corresponding peak ion concentration from 1543 nM +/- 312 nM to 1037 nM +/- 322 nM (P < .05); it also abolished the Ca(++) elevations seen in firmly attached platelets (gamma peaks). Blocking P2Y(12) had no effect on these parameters, and did not enhance the effect of inhibiting P2Y(1). Inhibition of phospholipase C had similar consequences as the blocking of P2Y(1), whereas inhibition of Src family kinases abolished both type alpha/beta and gamma Ca(++) oscillations, although the former effect required a higher inhibitor concentration. Our results demonstrate that, under elevated shear stress conditions, ADP signaling through P2Y(1) may contribute to the initial stages of platelet adhesion and activation mediated by immobilized VWF, and through P2Y(12) to sustained thrombus formation.
Asunto(s)
Agregación Plaquetaria/fisiología , Receptores Purinérgicos P2/metabolismo , Transducción de Señal/fisiología , Factor de von Willebrand/metabolismo , Calcio/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Adhesividad Plaquetaria/fisiología , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Estrés Mecánico , Fosfolipasas de Tipo C/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Platelet adhesion to von Willebrand factor (VWF) activates alpha IIb beta 3, a prerequisite for thrombus formation. However, it is unclear whether the primary VWF receptor, glycoprotein (GP) Ib-IX-V, mediates alpha IIb beta 3 activation directly or through other signaling proteins physically associated with it (eg, FcR gamma-chain), possibly with the contribution of other agonist receptors and of VWF signaling through alpha IIb beta 3. To resolve this question, human and GP Ibalpha transgenic mouse platelets were plated on dimeric VWF A1 domain (dA1VWF), which engages only GP Ib-IX-V, in the presence of inhibitors of other agonist receptors. Platelet adhesion to dA1VWF induced Src kinase-dependent tyrosine phosphorylation of the FcR gamma-chain and the adapter molecule, ADAP, and triggered intracellular Ca(2+) oscillations and alpha IIb beta 3 activation. Inhibition of Ca(2+) oscillations with BAPTA-AM prevented alpha IIb beta 3 activation but not tyrosine phosphorylation. Pharmacologic inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) prevented alpha IIb beta 3 activation but not Ca(2+) oscillations. Inhibition of Src with 2 distinct compounds blocked all responses downstream of GP Ib-IX-V under static or flow conditions. However, dA1VWF-induced responses were reduced only slightly in GP Ibalpha transgenic platelets lacking FcR gamma-chain. These data establish that GP Ib-IX-V itself can signal to activate alpha IIb beta 3, through sequential actions of Src kinases, Ca(2+) oscillations, and PI 3-kinase/PKC.
Asunto(s)
Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria , Transducción de Señal , Animales , Plaquetas/metabolismo , Plaquetas/fisiología , Señalización del Calcio , Humanos , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Adhesividad Plaquetaria , Proteína Quinasa C/metabolismo , Receptores de IgG/metabolismo , Familia-src Quinasas/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
We have identified a novel von Willebrand factor/fibrinogen/selectin-independent, platelet adhesion-promoting function of vascular PG-M/versicans that may be relevant in normal venous thrombosis and critical in atherosclerotic conditions. A purification scheme was devised to obtain vascular versicans, which by biochemical, immunochemical, and ultrastructural means were asserted to be 1) composed primarily of isoforms V1 and V2; 2) free of contaminants; 3) prevalently substituted with chondroitin-4-sulfate and dermatan sulfate (DS) chains; and 4) capable of binding hyaluronan to form link protein-stabilized ternary complexes. Real-time analysis of human platelet perfused under diverse shear forces showed that they largely failed to bind to several vascular and nonvascular proteoglycans (PGs). In contrast, they bound in a dose- and shear rate-dependent manner to vascular versicans, exhibiting a unique attachment-detachment kinetics and establishing a firm substrate tethering characterized with no significant aggregation. Digestion of these PGs with lyases and competition experiments with purified glycosaminoglycans revealed that platelet adhesion to vascular versicans was primarily mediated by their DS chains. Incorporation of the versicans into fibrillar collagen substrates augmented their adhesive activity and strongly promoted platelet aggregation at low and high shear rates. Affinity chromatography of platelet surfaces on DS columns identified a 120-140 kDa polypeptide complex that behaved as a specific vascular versican binding membrane ligand in solid-phase binding assays. These findings indicate that selective versican variants of the subendothelium may serve as ancillary GPIbalpha/integrin/selectin-independent platelet ligands in healthy and diseased vascular beds and may be directly responsible for the platelet accruing after rupture of atherosclerotic plaques.
Asunto(s)
Vasos Sanguíneos , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Adhesividad Plaquetaria , Agregación Plaquetaria , Animales , Aorta/química , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Bovinos , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/ultraestructura , Colágeno/farmacología , Dermatán Sulfato/metabolismo , Humanos , Cinética , Lectinas Tipo C , Proteínas de la Membrana/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , Isoformas de Proteínas/ultraestructura , Estrés Mecánico , VersicanosRESUMEN
We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca(++) concentration ([Ca(++)](i)) that are associated with sequential stages of integrin alpha(IIb)beta(3) activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibalpha (GPIbalpha) and the VWF A1 domain led to Ca(++) release from intracellular stores (type alpha/beta peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca(++)](i) oscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbalpha. Once adhesion was established through the integrin alpha(IIb)beta(3), new [Ca(++)](i) oscillations (type gamma) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbalpha-VWF bonds leads to alpha(IIb)beta(3) activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied alpha(IIb)beta(3), with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbalpha and alpha(IIb)beta(3) and lead to platelet adhesion and aggregation on immobilized VWF.