Biorheology
29 June 2006
Oslo Kongressenter, Norway
Chairman: J.J. Zwaginga, The Netherlands
Co-chairs: T. Diacovo, USA ; E. Grabowski, USA; M. Hoylaerts, Belgium;
Johan Heemskerk, Netherlands; G. Nash, UK(absent); Michael King, USA (absent).
In a overall well attended meeting (80 (start)- 20 (end) persons) several distinguished members of the Subcommittee and key-experts in the field gave presentations in 4 sessions.
Session 1: Activation of the coagulation cascade: the flow factor: moderator Johan Heemskerk. Jaap Jan Zwaginga on behalf of Kjell Sakariassen reviewed data on validating experiments with the developed ex vivo flow chamber set up (starting in the Basel Baumgartner period). He reported how these studies are performed under venous and normal and high arterial shear rates (resembling stenotic sites) over surfaces supporting platelet activation (collagen) or coagulation (tissue factor/ phospholipids). Efects of antithrombotic medication directed against platelets (cyclooxygenase or ADP receptor inhibition) vs, those influencing the coagulation (Warfarin or Xa, FVIIa inhitibion) are clearcut in terms of reduced thrombus formation or altered thrombus composition. The ex vivo experiments seemed to suggest that co-presence of collagen and (stenotic) shear induced platelet (procoagulant and thrombus) activation might need combinations of inhibitors to be effectively inhibited. A remark in the later discussion was that it is important now to compare these ex vitro flow data with those of flow chamber experiments in vitro.
Johan Heemskerk presented an overview of the requirements for rheological and flow experiments using whole blood to produce physiologically relevant results. He also discussed a number of practical considerations e.g. to work with small blood volumes. He further presented new data with animal and human blood, where thrombus formation on collagen was investigated under shear in the presence and absence of coagulation. In sum, he showed that thrombin took over the key role of platelet collagen receptors in thrombus formation under conditions of low (venous) shear rates, even in the absence of extrinsic coagulation activation. These results are promising in the development of standard flow protocols that incorporate coagulation.
Coen Hemker presented a model explaining under which conditions the enzymatic rate of thrombin formation or the diffusion of thrombin determines the effectivity of thrombin formation and the extent of coagulation. Diffusion is an important restricting factor in thrombin’s activity especially under conditions of flow. This implies that in venous arterial thrombi caveats are present where prothrombinase activity is rate-limiting, while at the thrombus surface diffusion is a rate limiting factor.
Part 2: Rheologic determinants in animal models studying hemostatic mechanisms: moderator Marc Hoylaerts . Imke Munnix presented perfusion data in which the stability of platelet aggregate formation was studied as a function of perfuming whole murine and human blood over surfaces of collagen. Whereas secondary perfusions with plasma maintained the integrity of preformed platelet aggregates, such perfusion with buffer led to gradual disaggregation and spreading of platelets over the collagen surface. Addition of ADP to the buffer could preserve the aggregate stability. Since these experiments were suggestive of active maintained platelet activation, after the initial formation of aggregates, further experiments were done in the presence of ADP receptor antagonists and in the presence of PI3-kinase inhibitors, selective for the different PI3-kinase isoforms. These perfusion studies showed that activation of PI3-kinase and was both required to explain the maintained platelet activation, required to keep the platelet integrin receptor IIb3 in a active configuration.
Barbara Furie compared two currently used models of thrombosis in the mouse, i.e. the laser-induced thrombus formation and the FeCl 3-induced thrombosis. The differences in the role of coagulatiin were discussed, as well as differences relating to the exposure of reactive collagen fibres, during platelet recruitment on injured blood vessels. A discussion ensued between the members of the biorheology subcommittee on this issue, included Dr. Barbara Furie. Thus, depending on intensity and length of exposure of vascular endothelium to laser light, the extent of injury can be mild to severe. Moreover, with FeCl 3, the thrombus can develop in a large area of the vessel wall, occupying and occluding the entire lumen of the vessel. In this case, there is a high probability that collagen is exposed. Standardization of both models is needed to permit comparison of results between labs and to understand the role of coagulation vs. platelet aggregate formation. Concerns were raised on whether laser-induced vascular injury leads to denaturation of proteins such as collagen, thus altering platelet adhesion in vivo. On the same note, a similar concern was raised about FeCl 2-induced vascular injury. Although antibodies to collagen have been used by the Furie lab to demonstrate collagen exposure in the latter case, concerns were raised on whether these antibodies can distinguish between the native vs. the denatured protein.. Possible methods to employ, during standardization efforts include exposing culture endothelial cells to laser light or FeCl 2 and determining the extent to which collagen is exposed. Such would require the development of antibodies that recognize collagen only in its native state, reagents that may not exist or are not readily available.
Part 3: Flow dependent effectivity of receptors and ligands: moderator Thomas Diacovo
Dr. Marc Hoylaerts reported on G allic acid modulation of platelet-leukocyte interactions: a model for flow dependent inhibitor efficacy and presented on the concept that phenolic compounds found in red wine such as Gallic acid can act as anti-inflammatories by impairing P-selectin mediated adhesion between leukocytes and vascular endothelium. Gallic acid (GA) is a major component of red wine (35-70 mg/L). To determine its role as possible anti-inflammatory, Dr. Hoylaerts evaluated the ability of this compound to inhibit P-selectin mediated-adhesion both in vitro and in vivo. Interestingly, results indicate that GA can mitigate this interaction but only under conditions of flow. The mechanism by which this occurs is still under investigation. Moreover, it does not interfere with the interactions between platelets and activated endothelium. This work demonstrates that GA in wine may contribute to the anti-inflammatory effects of red wine. Moreover, it demonstrates that certain P-selectin-inhibitors may only function under conditions of flow.
Thomas Diacovo reported on bond kinetic alteration in the interaction between vWF and platelets; effects on thrombus formation. The concept presented dealt with biophysical properties of the GPIb alpha – vWF-A1 bond that must be maintained within a narrow range, otherwise the ability to promote adequate hemostasis/ or form a thrombus will be perturbed. Examples of such alterations in humans include type 2M and type 2B VWD, where inclusion of single point mutations in the A1 domain of vWF decreases or enhances its interaction with GPIb alpha, respectively. By performing structure/functional analysis of the mouse recombinant A1 domain, Dr. Diacovo was able to identify a specific residue within this domain, the mutation of which reduced the lifetime of this interaction by >2-fold. Moreover, platelet accumulation on recombinant mouse vWF-A1 containing such a mutation was decreased by >60%. Using standard molecular techniques, this mutation was inserted into the mouse vWF gene. Animals were viable and displayed a bleeding phenotype distinct from that of the vWF KO animal. Collagen-induced aggregation and platelet adhesion to surface-immobilized collagen was perturbed in vitro. Moreover, laser-induced thrombus formation in the mouse cremaster muscle was impaired. This study demonstrates that the kinetics of the GPIb alpha-vWF-A1 bonds are optimally designed to function under the physiological conditions found on the arterial circulation and that perturbations in the properties of this bond can have devastating effects on hemostasis. In the subsequent discussion Dr. Farndale raised the issue on the ability of laser-induced vascular injury to expose collagen found in the ECM. Referring back to the results presented earlier by Dr. Diacovo’s which suggested this to be the case, while the work by the Furie lab showed the contrary, a discussion ensued. Points raised were the following:
Part 4: Standardisation issues and subcommittee proceedings: moderator Jaap Jan Zwaginga
- Richard Farndale presented a clear overview of the role of collagen as platelet activating (GPVI, α2β1), vWF binding in hemostasis and therefore in in vitro testing. He stressed the enormous variability of commercial preparates in their effectivity, which is caused by contaminants, covanent bond modification and differences in concentration and fibril size. Insights are fastly expanding in the confiding characteristics of this triple helical more or less crosslinked molecule with glycine –x –y triplet repeats and proline as x or y or hydroxyproline as y as being most efficient in forming a triple helix. In this respect, Farndale showed function analysis of synthetically engineered collagen peptides: mostly 9 triplet parts of the native collagens in conjunction with proximal and distal triple helix inducing glycine-proline-(hydroxyl-)proline triplet repeats. With this synthetic collagen peptide toolbox he showed that typical sequences with separate functions can be identified thus enhancing structure function insights on the collagen molecules and in vivo subtypes. Moreover (mixes of) these synthetic collagen related peptides (CRP’s) are now being considered as standard in vitro stimulus for platelet adhesion and activation/ aggregation in in vitro hemostatic tests.
- Jaap Jan Zwaginga closed the session stressing the improved biorheology network illustrated by the (new) members of the subcommittee (Hans Vink, Ton Lisman/ Peter Lenting, Shaun Jackson, Maurice Frojmovic, Scott Diamond, Patrick Andre, Arnaud Bonnefoy, Kjell Sakariassen, Philip de Groot, Richard Farndale). He furthermore gave an update on the 2 reports that were prepared as official short SSC publications in JTH with extended versions comprising extended references and tables to go on the SSC website. The present and future activities of the subcommittee were discussed and finally the subcommittee expressed the will to stimulate and effectuate synergistic work and exchange of thoughts with the animal study subcommittee for rheologic QC of animal models, the platelet physiology subcommittee in the area of flow dependent platelet assays and possibly the WP of the VWD subcommittee studying shear stress assays in VWD.