Jack J. Hawiger

Separation of human platelets from plasma proteins including factor VIIIVWF by a combined albumin gradient-gel filtration method using hepes buffer

Abstract Currently used gel filtration methods were investigated in order to obtain a system which would effectively separate platelets from plasma proteins, such as Factor VIII/von Willebrand factor (FVIIIVWF), in relatively small samples of human blood which are not amenable to repeated centrifugation. A new matrix, BioGel A-150 was tested and found to be equally as effective as Sepharose 2B for preparing gel-filtered platelets. However, it did not completely separate the FVIIIVWF as judged by ristocetin-induced platelet aggregation. Therefore, a stepwise albumin gradient was employed as a preceding step to gel filtration. Platelets obtained by this modification were not responsive to ristocetin and ADP unless human plasma, as a source of FVIIIVWF and fibrinogen, was added. Tyrode buffer, which shows variation in pH due to changes in pCO2, was replaced by the more stable zwitterionic HEPES buffer. The need for control of CO2 was thereby obviated, reactivity of platelets in vitro was maintained, and their functional life span was extended from 1 to 2 hours, This modification of existing methods allows efficient separation of human platelets from FVIIIVWF and from other plasma macromolecules in relatively small samples of blood with preservation of the functional and ultra-structural integrity of obtained platelet preparations.

Staphylococci-induced Human Platelet Injury Mediated by Protein A and Immunoglobulin G Fc Fragment Receptor

Bloodstream infections with staphylococci are accompanied by thromboembolic complications. We have studied the mechanism of the interaction of staphylococci with human blood platelets. Staphylococci that possess protein A, a bacterial receptor for the Fc fragment of immunoglobulin G (IgG), caused aggregation of human platelets in whole plasma accompanied by release of [(3)H]serotonin. These reactions were time and concentration dependent, requiring two or more staphylococci per platelet to give maximal response within 5 min. The interaction between staphylococci and platelets required the presence of cell wall-bound protein A and of IgG with an intact Fc fragment. It did not require an intact complement system. Cell wall-bound protein A (solid phase) was capable of aggregating human platelets in whole plasma. In contrast, free, solubilized protein A (fluid phase) did not cause measurable aggregation, and release of [(3)H]serotonin was reduced. An excess of free, solubilized protein A blocked aggregation of human platelets induced by staphylococci in whole plasma. The role of the Fc fragment of IgG in the staphylococci-human platelet interaction was demonstrated by an experiment in which free, isolated Fc fragment blocked aggregation of platelets in whole plasma induced by staphylococci. Furthermore, binding of (125)I-protein A to human platelets was demonstrated in the presence of complete IgG with intact Fc fragment but not in the presence of the F(ab)(2) fragment. Binding of the protein A-IgG complex to the human platelet Fc receptor was paralleled by the release of [(3)H]serotonin. These results represent a novel example of the interaction of two phylogenetically different Fc receptors, one on prokaryotic staphylococci and the other on human platelets. Their common ligand, IgG, is amplified by one Fc receptor (protein A) to react with another Fc receptor present on human platelets, which results in membrane-mediated aggregation and release reaction occurring in whole plasma. This mechanism can be of significance in the pathomechanism of thromboembolic complications at the site(s) of intravascular staphylococcal infection.

Human fibrinogen possesses binding site for staphylococci on Aα and Bβ polypeptide chains

THE fibrinogen molecule, which comprises 5% of plasma proteins, is involved in enzymatic reactions responsible for formation and dissolution of the fibrin clot1. Thereby, fibrinogen is being transformed by thrombin into fibrin, which is cross linked by glutaminyltransamidase (factor XIII), and ultimately degraded by plasmin. The Aα, Bβ and γ polypeptide chains (nomenclature according to the recommendations of the Committee on Nomenclature of the International Society on Thrombosis and Haemostasis, 1971) of fibrinogen involved in these reactions and the sites of enzymatic attack thereon are defined2–8. In addition to being involved in the enzymatic reactions responsible for formation and dissolution of the fibrin clot, fibrinogen exhibits the ability to interact in a non-enzymatic reaction with pathogenic staphylococci to cause their clumping9. Clumping of staphylococci is used to detect fibrinogen and its derivatives in blood and body fluids10,11. This reaction can detect as little as 0.03 µg human fibrinogen or its derivatives. The mechanism through which human fibrinogen interacts with staphylococci is however, unexplained and the localisation of the binding site(s) for staphylococci remains unknown. We therefore studied localisation of the binding site for staphylococci on human fibrinogen. We now report that the binding site for staphylococci is located on Aα and Bβ chains of human fibrinogen.

How to approach genome wars in sepsis

Sepsis continues to pose a clear challenge as one of the most difficult and costly problems to treat and prevent. Sepsis is caused by systemic or localized infections that damage the integrity of microcirculation in multiple organs. The challenge of sepsis and its long-term sequelae was addressed by the National Institutes of Health National Heart Lung and Blood Institute Division of Blood Diseases and Resources. Defining sepsis as severe endothelial dysfunction syndrome that causes multiorgan failure in response to intravascular or extravascular microbial agents, the National Heart Lung and Blood Institute panel proposed the concept of genome wars as a platform for new diagnostic, therapeutic, and preventive approaches to sepsis.