The views on the role of platelets in physiology and in pathogenesis have considerably changed in the recent past. While platelets had previously been seen only as contributors in primary haemostasis ...and as donors of negatively charged phospholipids to support thrombin formation, this view has had to be revised, at least since the discovery of specific receptors for coagulation factors on the platelet surface. Platelets are part of the body's immune defence system. They can interact with bacteria, pathogenic fungi and protozoa. The interaction of platelets with endothelial cells and leukocytes is crucial in innate and adaptive immunity. Platelets participate in the pathogenesis of the initial lesions and in the progression of atherosclerosis by inducing chronic inflammatory processes at the vascular wall, which result in the development of atherosclerotic lesions and atherothrombosis.
Acquired deficiency of antithrombin (AT), which in some patients could lead to thrombosis, has been a serious side effect of protocols which incorporate E. coli L-asparaginase (ASP) for the treatment ...of acute lymphoblastic leukaemia (ALL). In a longitudinal, prospective, non-randomized study children with ALL (n=27) were treated according to the protocol ALL-BFM-90. During the induction phase using prednisone, vincristine, daunorubicin and ASP, AT substitution was performed in 15/27 patients, when their plasma concentration decreased below 60% of normal with a concomitant increase of D-dimer formation. After the administration of the AT concentrate the patients, plasma concentration of AT increased and remained elevated after 18, 48, and 72 h. In addition, the plasma concentration of enhanced thrombin generation, D-dimer formation and plasminogen activator inhibitor 1 decreased towards normal levels. Although the observed laboratory findings may serve as evidence for a possible clinical benefit of AT substitution during ASP treatment, further randomized studies are requested to evaluate whether the use of prophylactic AT administration could reduce the incidence of thromboembolic events in childhood acute leukaemia.
The aggregation of platelets induced by collagens is considered an important step in primary hemostasis. Glycoprotein (GP) IIIb (GPIIIb, GPIV, CD36) has been proposed as a blood platelet receptor for ...collagen. Platelets from three healthy blood donors were shown to be clearly deficient in GPIIIb. These platelets aggregated normally in response to type I and III collagens. In addition, platelet factor 4, β-thromboglobulin, and adenosine triphosphate (ATP) secretion in response to type I and III collagens was normal. The findings indicate that GPIIIb is not the major, essential collagen receptor for type I and III collagens. This would explain why all individuals with GPIIIb-deficient platelets examined so far are healthy and, in particular, show no apparent evidence of hemostatic problems. However, in contrast to control platelets, no aggregation and impaired platelet factor 4, β-thromboglobulin, and ATP secretion was observed in response to type V collagen. Therefore, it is postulated that for type V collagen-induced aggregation both GPIa/lla and GPIIIb are essential.
Activated platelets provide a procoagulant surface for the assembly and expression of prothrombinase complex. Expression of activity is associated with the binding of the protease factor Xa (FXa) and ...the co-factor Va (FVa) to the procoagulant surface. A flow cytometric methodology to measure annexin V-FITC as well as FVa and FXa binding to ionophore A 23187 activated platelets is described. Annexin V-FITC was used to determine platelet exposure of phosphatidylserine. The binding was calcium-dependent and excess of unlabelled annexin V (10-fold) prevented the binding of the labelled protein. The binding of FVa and FXa to platelets was measured using specific FITC-labelled monoclonal antibodies. The FITC labelled antibodies were displaced by 10-to 20-fold excess of unlabelled antibodies. Binding was strictly Ca2+-dependent. Fixation of platelets by formaldehyde caused artificial binding of annexin V, FVa and FXa as well, irrespective of the platelet activation status. Using gel-filtered platelets, the binding of FVa increased with alpha -granule secretion but the amount of stored FVa was not sufficient to saturate the available platelet binding sites. Exogenous FVa was needed for maximal FVa binding to occur. No binding of FXa from internal platelet stores was observed. Addition of exogenous FVa and FXa resulted in FXa binding to the platelet surface. The methodology might be of use for the study of platelets from patients with bleeding disorders.
Platelet membrane glycoprotein IV (GPIV) is a cell-surface glycoprotein that has been proposed as a receptor for collagen. Recently, it has been shown that platelets with the Naka-negative phenotype ...lack GPIV on their surface, whereas donors with this phenotype are healthy and do not suffer from hematologic disorders. In this study, we compared Naka- negative platelets with normal platelets in adhesion to collagen types I, III, IV, and V and the extracellular matrix of endothelial cells (ECM) under static and flow conditions. No differences in platelet adhesion and subsequent aggregate formation on the collagens types I, III, and IV were observed under static and flow conditions. Adhesion of both homozygous and heterozygous Naka-negative platelets to collagen type V was strongly reduced under static conditions. Collagen type V was not adhesive under flow conditions. No difference in platelet adhesion to ECM was observed, which suggests that GPIV is not important in adhesion to subendothelium, for which ECM may serve as a model. These results indicate that GPIV is not a functional receptor for collagen under flow conditions.
Endovascular infection is a highly critical complication of invasive Staphylococcus aureus disease. For colonization, staphylococci must first adhere to adhesive endovascular foci. Von Willebrand ...factor (vWF) is a large, multimeric glycoprotein mediating platelet adhesion at sites of endothelial damage. Earlier it was demonstrated that vWF binds to and promotes the surface adhesion ofS. aureus, prompting this effort to identify the vWF adhesin. In Western ligand assays of S. aureus lysates, staphylococcal protein A (SPA) was recognized by purified vWF. Surface plasmon resonance demonstrated the binding of soluble vWF to immobilized recombinant protein A with a Kd of 1.49 × 10−8 mol/L. Using flow cytometry, the binding of fluorescein isothiocyanate–labeled vWF to S. aureus was found to be saturable and inhibitable by unlabeled vWF, antiprotein-A antibodies, or IgG. Isogenic Δspa::Tcr mutants were constructed by the insertion of a tetracycline resistance cassette intospa using allelic replacement, and it exhibited decreased binding of soluble vWF and decreased adhesion to vWF-adsorbed surfaces. The interaction was restored on complementation of the mutants withspa-containing plasmid pSPA7235. In conclusion, protein A confers interaction of S. aureus with soluble and immobilized vWF in a newly discovered function characterizing protein A as a novel member of the staphylococcal surface protein adhesin superfamily and suggesting its potential role in the pathogenesis of endovascular staphylococcal disease.
Abnormal platelet activation may be involved in prethrombotic states and lead to thromboembolism. When platelets become activated, they release thrombospondin (TSP) from their alpha-granules which ...binds mainly to the surface of activated platelets, platelet-derived microparticles and other blood cells. To determine bound as well as free TSP in a single assay, we developed an indirect ELISA to measure TSP in fixed whole blood. The intra-assay variance was less than 5% and 97% of purified standard TSP, added to whole blood samples, was recovered with the ELISA. Blood collected with a 20G needle into a syringe resulted in lower "whole blood TSP' values than blood collected with the Vacutainer system. Whole blood TSP levels were measured in 66 healthy blood donors (20F, 46M) aged 25-75 years. The mean whole blood TSP concentration was 33 +/- 19 ng/ml. No significant difference in whole blood TSP was found between healthy females and males (35 +/- 23 ng/ml vs. 33 +/- 17 ng/ml).
Glycoprotein IIIb (GPIV, CD36) has been proposed as the platelet receptor for thrombospondin (TSP). We found two healthy blood donors, whose platelets were shown to be GPIIIb deficient. These ...platelets expressed endogeneous TSP as control platelets and their binding capacity for exogeneous TSP was the same. These results indicate that GPIIIb is not the major TSP receptor on platelets. However, it is not yet possible to exclude that in GPIIIb-deficient platelets other proteins may substitute for GPIIIb in TSP binding.
Summary
A time-saving and sensitive method for the identification of antigens on unlabelled platelet glycoproteins (GP) based on immunoprecipitation has been developed. Platelet solubilisates
were ...incubated with antibodies to form GP-antibody complexes that were precipitated with Protein G Sepharose 4 Fast Flow (PGS). Pcs-bound material was eluted, separated by SDSPAGE under reducing conditions and visualized by silver staining. The method was designed as an alternative to radioimmunoprecipitation
for laboratories not equipped to work with radioactive substances. It is proposed as a complementary procedure for the characterization of platelet GP antigens by murine monoclonal antibodies and human alloantibodies.