Biorheology

July 18, 2002
13:00 to 17:00
Terrace Room
Boston Park Plaza Hotel

Chairman:  S. Diamond, USA
Co-chairs:  M. Hoylaerts, Belgium; G. Nash, UK; K. Preissner, Germany;
J.J. Zwaginga, The Netherlands

INTRODUCTION:

Dr. Diamond introduced the prevailing accepted assays in biorheology that allow study of adhesion on a surface (parallel-plate and tube flow) or bulk aggregation (cone and plate or concentric cylinder).   Flow forces are intrinsically important in understanding bonding function in thrombosis.  As an example, platelets have difficulty bonding to surface-sorbed vWF A1 domain while flow greatly facilitates bonding with a "hydrodynamic threshold" at ~100 1/s.      Additional examples of flow regulation of thrombosis were illustrated with fibrin formation around adherent neutrophils due to prothrombinase activity when Factor XIIa is not inhibited by corn trypsin inhibitor (CTI). CTI may see increasing use when working with recalcified whole blood or plasma in order to avoid unintended contact activation.  Membrane mechanics can play an important role in adhesion of real cells, e.g., platelet P-selectin-neutrophil PSGL1 bonding under flow where long membrane tethers can be pulled from the neutrophil to shield the bond from force loading.  Differences between bead systems and cells can often be fully reconciled by accounting for membrane deformation.  The additional point was made that recombinantly expressed receptors may not have the same kinetic/mechanobiology as intrinsic receptors.  Issues of sulfation and precise glycosylation structure are proving very important in the selectin literature. Adhesive interactions and mechanisms may depend on prevailing shear rates with respect to bonding pairs.   Dynamics of rolling to arrest and associated signaling may be shear dependent.  For example, at very low flow (< 100 1/s) normal RBC can form receptor mediated adhesion with activated platelets or neutrophils.   The exploration of the full flow range from static conditions to low shear (10 1/s) to higher shears (1000s 1/s) for surfaces ranging in low to high densities of adhesive ligands or cells provides a full characterization of bonding dynamics important to thrombosis.

As a summary, the subcommittee should be prepared to address emerging "omic" trends in (1) single bond mechanics (functional proteomics enabled by detachment assays, AFM, laser tweezer, micromanipulation studies); (2) cell adhesion under flow (functional cellomics or "hemodynamic phenotyping"); (3) integrative systems biology for combined study adhesion/coagulation/inflammation biology; and (4) high throughput assay (2D/MS, lab-on a chip, and "rheomics").

PRESENTATIONS:

Dr. Klaus Peissner
"The contribution of the urokinase receptor (uPAR) to the dynamics of inflammatory reactions."
An overview of uPAR interactions with uPA, vitronectin, b2-integrins sets the opportunity to study neutrophil uPAR regulation of Mac1 (CD18/11b) function in neutrophils.  Notably, in patients with acute myocardial infarction (AMI) but not angina or in healthy individuals, the level of leukocyte uPAR is elevated.  These cells are more adhesive to endothelium, an adhesion blocked by 20 µg/ml of anti-uPAR.  This adhesion was reduced by uPA.   In a flow assay (concentric cylinder), fMLP/Zn activated neutrophils were allowed to bind to fibrinogen coated beads.  Zn, which activates uPAR, caused a marked increase in heterotypic aggregation which was reduced by uPAR antibody.  This suggests a role for uPAR activation in elevating the avidity state of Mac1.  Platelets are an important source of Zn in the microdomains of a thrombus.  The D5 domain of kininogen, a cationic domain enriched in histidine, glycine, and lysine, has activity against the neutrophil-endothelium adhesion in a thioglycollate model.   GST-D5 protein or peptide 483-502 of D5 were anti-adhesive. D5 may reduce uPAR-Mac1 inteactions or uPAR-vitronectin interactions.  D5 increased the tail bleed time.  The role of uPAR as a Mac1 ligand on neutrophils during homotypic aggregation can be explored in the future.

Mony Frojmovic  (Past Subcommittee Chair)
Evaluation of shear-dependent drug efficacy on inhibition of platelet aggegation
Dr. Frojmovic highlighted the opportunity for evaluation of drug activity as a function of prevailing hemodynamic forces.  Notably, the IC50 of a drug may change depending on the prevailing flow.  The time-dependent, binding affinity of fibrinogen for active GPIIbIIIa was a focus of study and may be shear dependent.   This phenomenon, long observed with platelets, can be observed with purified IIbIIIa on beads where bound fibrinogen is easily desorbed with Ro44883 when the drug is added early on, but after 20-30 min fibrinogen is not easily displaced by Ro.   The conclusion is that fibrinogen can go deeper into the binding pocket of IIbIIIa after a period of about 30 minutes.  Under shear conditions, however, Ro disaggregated fibrinogen-GPIIbIIIa interactions, regardless of the age of the aggregates.  The conclusion is that under flow conditions, the fibrinogen cross-bridges may not be able to access the deep pocket, irreversibly bound state.  Again the equilibrium properties of the binding interaction in a biacore or binding assay with zero force (other than thermal motions) may not be predictive of performance of a system under hemodynamic forces.

Scott Diamond
Normal erythrocytes: Not so passive and a little sticky during coagulation
Dr. Diamond noted that DVT is a low shear pathology whose prevention includes vascular compression therapy to elevate blood flow. A study at depressed venous flow conditions under 100 1/s indicated that normal RBC can adhere to collagen adherent platelets via GPIb and CD36 mechanisms, but that this adhesion was not blocked by antibodies against IIbIIIa, TSP, vWF, or Pselectin.  RBC-platelet adhesion was blocked by fibrinogen or EDTA.   RBC also adhere at these low flow conditions to fMLP-activated neutrophils through a CD1811b mechanism.  One of the RBC mac1 ligands was found to be ICAM4 (LW antigen).  The other ligand is not known.  RBC were found to bind under low flow conditions to fibrin formed from plasma.  

Jaap Jan Zwaginga
Platelet adhesion decreases to lysed fibrin whereas neutrophil adhesion remains intact under conditions of flow
Dr. Zwaginga discussed neutrophil receptors (PSGL1, ESL1, Lselectin and Mac1, LFA1, and VLA4) in the context of endothelial counter-ligands (Pselectin, Eselectin, CD34/Madcam/sLeX, and ICAM1 and VCAM1).  While neutrophils had little adhesion to endothelial matrix (200/mm2 which drops to near zero adherence at about 300 1/s), neutrophils can adhere to ECM in the presence of platelets (1000/m2 which drops to low levels at about 800 1/s.  Neutrophils were noted to roll on surface spread platelets, roll and arrest on platelet/fibrin, and arrest on fibrin alone (at low shear rate < 100 1/s).   The platelet ICAM2-neutrophil LFA1 interaction was noted to be fairly minor.  Accumulation of neutrophil strings/clusters on fibrin was found to be dependent on Lselectin, presumeably via Lselectin-PSGL1 neutrophil-neutrophil mediated capture.  Neutrophils also adhere better to fibrin that is aligned in the direction of flow, via improved cell-fibrin contact and not via a new epitope in aligned fibrin fibers.  Finally it was observed that neutrophils adhere nicely to partially degraded fibrin (tpa + plasminogen) while platelet adhesion is lost.  This may have importance to clot remodeling and wound healing.  The mac1 binding epitiopes on fibrin gamma chain are preserved during fibrinolysis.  

Marc Hoylaertz
Shear controlled platelet rolling over vWF is blocked by antibody shielding and by site directed mutagenesis of the A1 domain binding site for platelet GPIba
Dr. Hoylaertz explored the function of the GPIb-vWF interaction under flow conditions.  This interaction, once thought to be long lived, is now recognized to be a short lived bond with a life under 1 sec under shear conditions.   A number of loss of function mutants of the A1 domain were explored with special emphasis on conserved amino acids between 563 and 622.   Binding to wt A1 was found to increase with shear stress. Antibody AjvW2 appears to bind position 563, 566, 570 which are on the binding face of A1.  AjvW2 competes with heparin for these sites on A1.   Notable loss of function mutants were D570A and R616.   Notable changes in platelet attachment trends with shear rate (200, 1000, 1500 s-1) were noted with these mutants.  Videos of rolling demonstrated fast rolling and transient short lived translocation on these mutants that have loss of function.  A full analysis of rolling velocity, pause distribution, and catch efficiency will help define the mechanical properties of the binding face as a function of bond loading. These issues are critical in understanding platelet recruitment under arterial flow conditions and the role of heparin as an antiadhesive.

RECOMMENDATIONS:
  1. The committee will review and submit a report providing guidelines for the interpretation of single-molecule bond mechanics in the context of thrombotic/inflammatory reactions under hemodynamic conditions.  This includes a summary of kinetic data and their analysis to provide kinetic parameters.
  2. The committee will review and submit a report providing guidelines for the interpretation of pharmacological agents (IC50) as a function of hemodynamic environment.
  3. The committee will highlight emerging trends in lab-on-a-chip, miniaturization, systems modeling, and proteomics that relate to functional phenotyping of blood function under hemodynamic conditions.