Professor Yasuo Ikeda presented the final working party report on "Effects of Flow on Endothelial Cell (EC) Functional Activities" cochaired with Prof. Kjell Sakariassen. He described the various factors affecting platelet function that will be subject to modulation by flow effects: PGI2 and NO, both increasing with shear stress, associated with decreased platelet aggregation; tPA increase and PAI-1 decrease, with elevated fibrinolysis; tissue factor (TF) decrease, and thrombomodulin ™ increase, associated with decreased coagulation; all the changes generally associated with anti-thrombogenicity associated with increasing shear stresses. The synergistic effects of cytokines (IL-1 and TNF) with shear stresses on tPA and PAI-1 were described at shear stresses of 12-24 dynes/cm2. In addition, shear stress decreased TNF expression in HUVECS. Flow device most useful was the cone-in-plate microcouette.

Prof. Jeff Hubbell gave his final working party report, cochaired with Prof. Steve Hanson, on "Local Drug Delivery and Flow." He discussed the issues of transport and biorheology associated with the delivery of drugs to the artery wall, and to thrombi adherent to the arterial wall. He outlined the methods best used to date, including the use of a) permanent implants such as graft sleeves, b) polymer depots, either within the surrounding adventitia, within the media, or within the artery lumen, and c) a variety of catheter designs, with and without imposed pressure or electric fields. He described some distinct chemical structures designed to promote drug deliveries, including bilayer depots where the drug-loading gel is covered by a polymer gel with low permeability not containing any drug, and the use of heparin binding of growth factors for delivery devices .

Prof. Mony Frojmovic gave a final working party report on "Blood Cell Aggregation in Flow."

Both biological and biophysical issues associated with methodological approaches and data interpretation were described. The biological issues included the presentation of new receptors (e.g, P-selectins and activated GPIIbIIIa), exposure to new agonists, differential importance of newly secreted ligands that remain platelet surface-bound versus soluble ligands, multiple cross-associations of receptors and ligands, the time between cell activation and actual cell collisions, as well as the ‘age’ of the activated cell in circulation, and activation of intracellular signal transduction. Biophysical issues include the effects of flow on collison frequency , adhesion and de-adhesion related directly to surface molecules present on platelets and other blood cells at the time of actual collisions. The choice of flow devices, and model surfaces or model spheres in suspension, is critical to proper and biologically meaningful evaluation of the molecular biorheology of platelet-platelet and heterotypic blood cell collisions, best done with particles in suspension in couette flow. Parallel plate flow devices, on the other hand, are more appropriate for studies of platelet- thrombus collisions. The build-up of local metabolites with time in ‘closed’ devices must be properly evaluated, quite possible with rapid measurements of shear-associated aggregation within seconds of cell activation versus longer incubatin times prior to shear. Possible effects of hydrostatic pressure on cell function must also be examined.

Prof. Mony Frojmovic introduced the symposium on " Rheological Bedside Devices for Monitoring and Managing Vascular Diseases" with a brief overview of current status and future needs. He discussed biorheological issues in the design of flow devices for clinical monitoring of thrombosis and coagulation, and the relation between platelet biology, flow and the choices of anti-thrombotic drugs. He also described the need for relevant in vitro flow-dependent test for monitoring drug efficacy.

Prof. Don Gabriel presented a brief overview of the clinical issues in monitoring the thrombotic state, as a complementary introduction to the use of bedside clinical devices. He reviewed flow effects on rheopheresis of platelets concentrating near vessel walls; effects of flow on fibrin organization; and some of the molecules regulating platelet adhesion to collagen and fibrin.

Prof. Marc Hoylaerts discussed the use of rectangular microcapillaries for the study of platelet deposition onto well-defined surfaces, including seeded endothelial cells (EC); studies of leukocyte-EC interactions are also readily made with this approach. Quantification is performed with videomicroscopy and computer interface. He described the use of anticoagulated hamster blood evaluated between 300 and 2700/sec shear rates. Good platelet deposition was observed onto fibrin, vWF or collagen I —coated glass surfaces in the rectangular micro-capillaries. Anti GPIb ‘drugs’ inhibited hamster platelet deposition onto collagen I by 50% at 1300/sec, but anti-GPIIbIIIa drugs were without effect; whereas at 2700/sec, anti GPIIbIIIa drugs potentiated, platelet surface coverage, while anti-GPIb drugs were without effect .

Critical factors that need to be considered in choosing a flow system include: blood volume; shear stress; parameters measured such as closure times and cell rolling; mono versus re- perfusion; reproducibility and sensitivity of the system; ease of use; and evaluation of anti-platelet drugs. The rectangular capillaries are good for low volume, but not practical for bedside assays at this time, largely due to need for ‘unstable’ protein-coated surfaces. They are, however, very useful because of direct visualisation of platelet deposition via videomicroscopy. Dr Sakariassen pointed out the importance of eventually testing fresh non-coagulated blood in platelet function testing.

Prof. Yasuo Ikeda described the use of a quartz crystal microbalance for the evaluation of platelet deposition onto protein-coated surfaces. In spite of problems in analysing the cellular overlayers with standard equations applicable to molecular reactions, due to viscoelasticity of the cells, quantitative analyses were feasible and provided real-time measurements of platelet deposition under various pathological conditions. The usefulness of this system for monitoring the time course of binding of liposomes to vWF-coated surfaces was well demonstrated with liposomes containing different densities of rGPIbalpha, as well as liposomes containing GPIa-IIa binding to collagen.

Dr. Eric Heilmann, from Dade-Behring, provided a detailed overview of the PFA-100 flow device, which is based on the closure of a capillary with a collagen or ADP-Adrenaline coating. The clinical usefulness of this device was described for a variety of situations, including von Willebrand’s disease, aspirin-induced bleeding, and Glantzmann’s thrombasthenia. A new, very important observation on the two patient populations with distinct response to treatment with the anti-GPIIbIIIa drug, Reopro, observed 19 hours after administration, was described: about 1/3 of patients returned to normal, with 2/3 still showing abnormal closure times.

Dr. Bruce Lages, on behalf of Dr. Conan Li, from Xylum, gave a detailed outline of the Clot Signature Analyser. The instrument generates an orifice with flow through both the orifice and a channel containing a collagen fiber. Native blood is used, with both platelet function and clotting times readily monitored. The platelet-mediated hemostasis time (PHT) reflects mainly platelet adhesive function, with no effects of fibrinogen/fibrin, while the collagen-induced thrombin formation (CITF) time reflects fibrin formation actually visualised in the platelet fibrin-rich thrombi formed. Useful data from a number of clinical situations were reviewed. In particular, low molecular weight heparin had more effect on PHT, likely related to thrombin formation affecting platelet function and not fibrin formation. Two anti-thrombotic drugs showing total block of platelet aggregation in aggregometry showed some rebound effects in analyses of the blood with the CSA analyser, paralleled by measurement of platelet-bound fibrinogen by flow cytometry.

Dr. David Varon, from the University of Tel Aviv and Tel Hashomer Hospital, presented results on the novel use of a micro cone-in-plate device for the assay of platelet adhesion and aggregation with anticoagulated whole blood from patients. Bedside data output is provided by computer interface analysis of platelet surface coverage and aggregate size. The relative usefulness of native polystyrene versus ECM-coated plastic was described. A number of clinical situations were described which could be ‘characterised’ from changes in these platelet parameters.

Dr. Robert Hillman, from Accumetrics, described the use of fibrinogen-coated beads in a bedside device for monitoring blood drug concentrations for those directed at the GPIIbIIIa platelet receptors. Of the more than 300,000 patients per year in N.A receiving Reopro, about 60% are getting sub-optimal treatment, with high variability of reopro concentrations in patients’ blood leaving hospitals, with observation that less than 80% GPIIbIIIa blockade associated with much higher event rates in cardiovascular disease. Blood drug monitoring and receptor blockade measurements will become increasingly important with the generation of increasing numbers and types of antithrombotic drugs.

Dr. Marc Carr, from Hemodyne, described a bedside device for monitoring clot retraction and fibrinolysis. Following in vitro addition of thrombin to a patient’s blood, the mechanical properties of the fibrin network and the contractile forces generated by the platelets are measured. He described the very different sensitivities seen for effects of reopro on whole blood aggregation with 80% inhibition versus effects on clot retraction associated with maximal platelet activation due to use of thrombin for the observation. The high interdonor variabilities in responses for clot retraction were noted. Some impressive examples of clinical situations showing altered clot retraction and fibrinolytic capacities were described.

Each of the methods developed and described above has been used clinically and calibrated in standard platelet-defective states and coagulopathies.

Prof. Gordon Lowe, in collaboration with Prof. J Danesh, told the gathering to attend an afternoon subcommittee session as there was no time to give a brief final report on "predictive values of rheological variables for vascular events," focusing on measurable changes in fibrinogen concentrations as a likely marker for acute phase reaction associated with vascular events. A written report will however be submitted to our committee in the coming months.

The chairman solicited suggestions for new topics for future meetings, and specifically for the Maastricht meeting in June of 2000, which include: 1) a follow-up symposium on ‘bedside devices," possibly jointly with other subcommittees such as the platelet physiology group; 2) Rheological Models for Bleeding/Thrombotic Disorders; 3) flow effects on cell structure-function; 4) what makes arterial blood physiology so distinct from that of venal?

Attendance was excellent with 115 participants .

P.S. Any ICTH attendees wishing to become ‘consulting members’ of our Biorheology subcommittee are invited to e-mail the chairman at: mony@med.mcgill.ca.