SCIENTIFIC SUBCOMMITTEE SESSION
6 July 2007 Palexpo, Geneva, Switzerland

Biorheology

Chair: J.W.M. Heemskerk (The Netherlands)
Co-Chairs: T. Diacovo ( USA), E. Grabowski ( USA), M. Hoylaerts ( Belgium), M. King ( USA), G. Nash ( UK), J. Zwaginga (The Netherlands)

The Biorheology session was an intensive meeting with 13 presentations divided into 4 parts, corresponding to two working parties and two special projects. The session was well attended by about 190 participants throughout.

Introduction
Chairman Dr. J. Heemskerk presented a short overview of the activities of the Subcommittee on Biorheology in the past year. He continued with an overview of the working parties and special projects of the subcommittee, as also reflected in the program of this year’s session.

WP1: Practical biophysics of cellular bond characteristics mediated by flow (chaired by T. Diacovo and M. King)
Dr. T. Diacovo reiterated the mathematical basis to describe bond characteristics: from on-off rates and affinity state description, equilibrium kinetics to modern ( Bell) modeling of dynamic disruption of chemical bonds by mechanical (shear) force. By the use of a glycoprotein Ib (GPIb)-coated microspheres in interaction with wildtype and von Willebrand disease (VWD) type IIb peptides of VWF A1 domain, Dr. Diacovo presented experimental data that validated the new mathematical modeling system. Mutant VWF A1 domains were engineered on the basis of expected changes in their binding to GPIb. Adhesion changes could be shown in the microsphere system, and be quantified as specific changes in bond life time and Kd using this modeling approach. The in vivo relevance of the modified A1 domains was furthermore shown in a mouse knock-in models. Dr. M. King subsequently showed advances in mathematical modeling of the platelet tethering to VWF and the platelet aggregate formation. Using the Bell model, determinants like shear, platelet and glycocalyx dimensions and GPIb density, he presented algorithms that describe dynamic interactions in 3D of multiple platelets with the vessel wall and with each other. These algorithms are experimentally validated for platelets at lower shear rates, but in the future additional complexity will be added, e.g. shear induced platelet activation, signal transduction, and affinity changes of surface receptors.

WP2: Flow-determined modulation of molecular processes in thrombosis and haemostasis (chaired by J. Zwaginga and K. Sakariassen)
Dr. A. Bonnefoy demonstrated novel results and techniques that in addition of the A3 domain also the A1 domain of VWF is involved in the binding of VWF to collagen. This binding is inhibited by heparin, suggesting that anticoagulation with heparin can also interfere in platelet-VWF-collagen interaction. This recognition is of immediate importance for the attempt in WP2 to further characterize patients with aberrant VWF, being recognized as VWD patients, using flow-mediated assays. The penetrance of flow-incorporating assays in the clinical routine, however, is limited. Dr. J. Eikenboom gave an overview of the current way of classification of VWD patient, such as recently agreed by the SSC on VWF. In most types of VWD (type 1, types 2ABNM, and type 3), patients display a variable extent of bleeding. In addition to the antigen level of VWF, also qualitative abnormalities of VWF are instrumental to the diagnosis. Dr. Eikenboom concluded that assays which incorporate flow conditions might well increase diagnostic sensitivity in regard of clinical symptoms. Dr. J. Zwaginga followed up on this subject and reported on a currently reviewed position paper of the Subcommittee on Biorheology, stating which and how flow assays could best contribute to further explaining remarkable phenotypes of VWD patients. Although cone-and-plate analyzers are commercially available, perfusion chamber-based assays because of their extensive validation in VWD variants, are regarded as most promising in this respect. Several modifications in these assays, however, should be dealt with before clinical validation and prospective multi centre trials can be set up. For the latter, collaboration with the SSC Subcommittee on VWF is indispensable and will be investigated in the coming year.

SP1: Standardization thrombogenic surfaces in flow: potential of synthetic surfaces (chaired by E. Grabowski and J. Heemskerk)
On behalf of Dr. R. Farndale, Dr. N. Pugh presented the methodologies – type of flow chamber, staining procedure and confocal microscopic imaging – used to measure thrombus formation in vitro under flow conditions. Novel triple helical collagen peptides, designed for binding to glycoprotein VI (GPVI), integrin alpha2beta1 and VWF, are synthesized by this group and used as an adhesive surface for aggregating platelets. Dr. Pugh showed that the presence of all three motifs is required for optimal thrombus formation at high shear rates. He discussed the various ways of quantification of this process, and the importance of a standardized coating procedure for reproducible results. Dr. K. Sakariassen gave an overview of results from ex vivo perfusion studies, where non-anticoagulated human blood is flowed over human type III collagen. He stressed the importance of quality control of the collagen preparation and also of the way of application of collagen on the surface (e.g., spraying). He underlined the importance of thrombus volume as an endpoint parameter, and he pointed to the phenomenon of axial dependence of thrombus formation. The reproducibility of thrombus formation over a 10 year period appeared acceptable, particularly in studies where only non-smoking healthy individuals were included as blood donors. As the last speaker in this session, Dr. J. Cosemans compared the process of flow-induced thrombus formation on various types of adhesive surfaces While the thrombotic process on plaques was found to be variable from one plaque to another, it was more standard on purified collagen type I preparations. She presented evidence that the degree of processing of plaque-derived collagen by matrix metalloproteinases contributes to the variable results obtained with plaques. On the other hand, synthetic triple-helical peptides with collagen-based motifs for adhesion to alpha2beta1 or GPVI, alone or in combination with VWF gave highly promising results in achieving reproducible thrombus formation under flow. These results are promising for the generation of artificial adhesive surfaces to check for the activity of specific adhesive platelet receptors.

SP2: Models of thrombus formation in vitro and in vivo (chaired by M. Hoylaerts and G. Nash)
Dr. S. Jackson gave an overview and showed examples of the advantages and pitfalls of in vitro and in vivo models to study thrombus formation. He demonstrated the great potency of microcapillary flow chambers in combination with high-resolution confocal microscopy and other recently developed multi-imaging systems. He showed how total internal reflection fluorescence microscopy can be used to get detailed insight into the adhesive properties of single platelets subjected to flow. Dr. Jackson then focused on the additive contributions of immobilized collagen and VWF in platelet adhesion and activation. Comparable in vivo studies, however, seem to indicate that platelets adhering to the vessel wall are in a lesser activated state than is anticipated from the in vitro flow studies. Various factors appear to mediate thrombus instability in FeCl3 in vivo models (which are partially different from the in vitro situation), i.e. the vascular substrate, the local rheology, the activation state of the endothelium and the contribution of thrombin. Dr. L. Brass presented evidence for novel mechanisms that regulate thrombus growth and stability. He discussed current insight into the factors determining platelet-platelet interaction in a laser-induced vascular damaging model. Particularly studies with genetically modified mice have given support to the idea that the thrombotic process consists of three phases: a phase of rapid platelet accumulation, a slower growth and plateau (RGD insensitive) phase, and a gradual declining phase. Mice with mutated G12, lacking semaphorin 4D, or lacking ESAM show different profiles of thrombus formation, in which one or two of these phases are altered. Such modeling is likely to help understanding the multi-molecular interactions that are involved in the thrombotic process.

Dr. G. Nash gave an overview of the blood rheological and haemodynamic factors influencing platelet deposition in various flow models. He stressed the importance of margination of cells to the outer part of the vessel wall (determined by haematocrit and red cell aggregation). This results in blunting of the velocity flow profile, which for example is different for horizontal and vertical vessels, and for thin and thick vessels. The last speaker, Dr. E. Grabowski, informed on the factors determining the adhesion of platelets to cultured endothelial cells in flow. Treatment of the cells with alpha-shiga toxin and factor VIIa or TNF-alpha caused massive adhesion of strings of platelets, as a consequence of the activation of endothelial cells. This adhesion was tissue factor-dependent. Dr. Grabowski informed on the measurement method, the adhesive receptors involved and on the pathological conditions at which this platelet adhesion is relevant.