Invited Talks: T. Diacovo (USA), M.B. Lawrence (USA)

Professor Scott Diamond opened the session with introductory remarks noting the emergence of tools to investigate single receptor-ligand bonding kinetics under applied mechanical loads such as hemodynamic shear forces. Quantitative kinetic and mechanical descriptions of bonding and debonding of individual receptor-ligand chemistries during homotypic and heterotypic interactions provide a starting point for analysis of cell mediated coagulation under hemodynamic forces. Studies of individual chemistries is required before multiple receptor-ligand pair interactions in the contact area can be defined from an adhesion strength perspective.

Symposium:

Professor K. Preissner presented an overview of neutrophil-endothelium and neutrophil-matrix interactions with a focus on CD11bCD18 (Mac-1). Using a thioglycollat injection to the peritoneum as a inflammation/extravasation model in uPAR(-/-) mouse followed by peritoneal lavage at 1-4 hr, a 30 to 50 % decrease in neutrophils in the KO mouse was noted relative to wt mouse. Also, large decreases in eosinophil and monocyte levels were noted in the KO mouse. This suggested an important role of uPAR interactions with Mac-1 where uPAR may modulate the avidity of Mac-1. uPAR may exert a "preactivating" effect on beta2-integrins without effect on beta2-integrin antigenic levels on the cell surface. In static adhesion assays, the interaction of uPAR with vitronectin was enhanced by zinc ion and uPA. It was noted that free zinc ion exists in platelet granules and can be released at sites of thrombosis. Only 1 to 2 % of the 50 uM total zinc in plasma is considered free. The role of domain 5 of kininogen especially H475-486 binds uPAR and interferes with uPAR preactivation of Mac-1. The effect of flow on local zinc ion at the site of local platelet adhesion remains to be determined.

Professor M. Frojmovic presented an overview of platelet surface receptors for vWF, fibrinogen and thrombospondin (TSP-1). Importantly the alpha granule contains fibrinogen, TSP-1, fibronectin, vWF, 2b/3a (20,000 copies), and P-selectin. Release of granules by washed platelets in buffer is sufficient to provide fibrinogen and vWF for aggregation in flow conditions. Measurement of initial singlet consumption in a sheared suspension of platelets in a couette viscometer allows determination of the collision efficiency which typically ranges from 0.01 to 0.3. Using beads for presentation of individual ligand to the platelet allows probing of specific receptor-ligand interactions. It was convincingly shown that resting platelets have no binding interaction to fibrinogen immobilized to the bead surface. Activated platelets are captured by fibrinogen presented by immobilized 2b/3a beads with an efficiency of about 0.1. Interestingly, beads with immobilized active 2b/3a can bind fibrinogen which in turn binds TSP, allowing the creation of a remarkable 169 nm linkage between beads.

Professor T. Diacovo demonstrated using recombinant A1 domain of vWF that the platelet GPIb bond with A1 domain lasts for under a second when subjected to hemodynamic forces. Values of the dissociation rate of about 5 s-1 indicate that the bond is short lived. Additionally, work with the 2B mutant A1 domain showed that the GPIb-2B A1 domain interaction was about 10 times longer lived. Furthermore, a critical level of shear stress was needed (about 1 dyne/cm2) to maintain platelet rolling on A1 domain. Platelets, at shear stresses above 1 dyne/cm2, maintained stable rolling which was rapidly destabilized when the shear stress was dropped below 0.3 dyne/cm2. This rapid reversal of adhesion with a decrease in flow was mediated by a monomeric A1 domain that was physisorbed or biologically linked to the surface. This finding suggests that flow does not effect the A1 domain. Placement of the A1 domain on beads suggested that the dissociation process of GPIb-A1 is very similar to that of P-selectin-PSGL-1.

Professor M. B. Lawrence presented work on the membrane deformation of neutrophil microvilli during rolling on selectin coated surfaces. Beads coated with PSGL1 and fixed neutrophils had very similar bond breakage kinetics as described by the Bell Model,however, unfixed neutrophils had different kinetics for the breakup of the bonds. This was shown to be due to the extension of membrane tethers which were pulled at longer lengths at higher shear forces. These tethers were clearly detected by SEM as well as by DIC light microscopy. The tethers are pulled as viscoelastic elements for short periods of time and then undergo viscous deformation. Extension of the tether enhances the lever arm and thus reduces the forces experienced by the bond. Membrane tethers thus shield the bond from loading and enhance the bond life. The Bell model remains the standard for analysis of force induced debonding (koff = koff(0) exp (r Fb/Kb T) where koff(0) is the zero stress off-rate, r is the reactive compliance, Fb is the force on the bond, Kb is the Boltzman constant, and T is the temperature. Membrane deformation alters lever arms which in turn changes Fb, the force on the bond.

Professor S. L. Diamond explored the role of neutrophils in fibrin formation. Neutrophils adherent to fibrinogen coated surfaces led to massive fibrin deposition after 15 min of perfusion of recalcified PFP. This deposition was substantially reduced with anti-b2 antibody and by corn trypsin inhibitor to block XIIa pathways triggered by contact activation. Almost no fibrin was detected after 15 min perfusion of CTI-treated recalcified plasma over neutrophils adherent to fibrinogen. Similarly, no fibrin was detected after 15 min perfusion of CTI-treated recalcified plasma over platelets adherent to fibrinogen. In contrast, large amounts formed under these conditions when CTI-treated plasma was perfused over p/n mixtures adherent to fibrinogen. This fibrin formation was reduced by inhibitors against elasase or cathepsin G. This suggested that neutrophils and release proteases that activate platelets that are spread on fibrinogen. It was noted that Dacron graft models where fibrin deposition is reduced by anti-p selectin may be due to the fact that the Dacron promotes fibrinogen deposition in the absence of collagen and neutrophils may help cause greater levels of platelet activation after their spreading on fibrinogen as indicated by PS, calcium, and prothrombinase activity.
 
 

Professor M. Hoylaerts gave an overview of monocyte-platelet interactions as well as the role of monocyte-tissue factor in stirred blood coagulation. Through measurements of thrombin activity it was determined that WBC-platelet interactions were important for thrombin generation in a rotated plate assay.

SUMMARY

The Subcommittee will help to standardize receptor-ligand assays that seek to define single bond mechanics in cellular and cell-mimic systems under flow conditions that are physiologically relevant. With definition of individual bond dynamics, it may become possible to understand cell contact area dynamics where 3-5 different bonding mechanisms are holding the cells together. Further emphasis will be given to phenotypic performance of blood components in the context genotyping and phenotyping chip and microarray data.