ABSTRACT
GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.
Subject(s)
Antibodies, Monoclonal/pharmacology , Platelet Adhesiveness/drug effects , Platelet Glycoprotein GPIb-IX Complex/immunology , von Willebrand Factor/pharmacology , Animals , Antibodies, Monoclonal/isolation & purification , Blood Platelets/chemistry , Blood Platelets/drug effects , Blood Platelets/immunology , Drug Interactions , Epitopes/chemistry , Epitopes/immunology , Humans , Male , Mice , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIb-IX Complex/physiology , Protein Binding/drug effects , Protein Structure, Tertiary , Rats , Species Specificity , von Willebrand Factor/antagonists & inhibitors , von Willebrand Factor/metabolismABSTRACT
Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.
Subject(s)
Platelet Activation , Platelet Glycoprotein GPIb-IX Complex/metabolism , Thrombosis/blood , Animals , Antibodies, Monoclonal , Antithrombin III/biosynthesis , Antithrombin III/drug effects , Biomarkers/blood , Blood Platelets/drug effects , Coagulants/metabolism , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Epitopes , Flow Cytometry , Pancreatic Elastase/metabolism , Peptide Hydrolases/biosynthesis , Peptide Hydrolases/drug effects , Platelet Factor 4/biosynthesis , Platelet Factor 4/drug effects , Platelet Glycoprotein GPIb-IX Complex/immunology , Platelet Glycoprotein GPIb-IX Complex/physiology , Platelet Membrane Glycoproteins/immunology , Platelet Membrane Glycoproteins/metabolism , Platelet Membrane Glycoproteins/physiology , Rats , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/physiology , Sensitivity and Specificity , Solubility , Thrombin/biosynthesis , Thrombin/metabolism , Thrombin/pharmacology , Thromboplastin/administration & dosage , Thromboplastin/pharmacology , Thrombosis/metabolismABSTRACT
Real-time biospecific interaction analysis based on optical detection by surface plasmon resonance was used to develop an accurate one-step method for the direct measurement of free protein S in human plasma. This assay was validated, compared with classical immunological methods and shown to be suitable for the routine clinical diagnosis of protein S deficiency. The method relies on the specific capture of free protein S directly from plasma by a monoclonal antibody (mAb), 34G2, immobilized on a sensor chip surface. A calibration curve was established with serial dilutions of standard plasma (working range 5-50%) and a linear relationship was found to exist between the relative response in resonance units (RU) and the concentration of free protein S expressed as percentage plasma dilution (r = 0.99). The specificity of the assay was confirmed using purified human protein S and polyethylene glycol treated plasma. In addition, it could be demonstrated that no dissociation of C4b-BP-protein S complexes occurred under the chosen experimental conditions. The technique was reproducible with inter-assay, intra-assay and inter-sensor chip variation coefficients of 1.5-5.4%, 2-3.1% and 4.4-4.9%, respectively, as evaluated in two different plasma samples. Since all tests are automatic and long series of analyses can be performed with the same sensor chip, the method was applied to the determination of free protein S antigen in plasma from 20 normal blood donors and 38 thrombophilic patients. Results displayed excellent correlation with those of free protein S enzyme-linked immunosorbent assay (r = 0.99) and rocket immunoelectrophoresis of polyethylene glycol-treated plasma (r = 0.93).
