Biorheology

July 12, 2003
09:00 to 13:00
Hall Sonata (Hyatt)
The International Convention Center, Birmingham

Chairman:  S. Diamond, USA
Co-chairs:  M. Hoylaerts, Belgium; G. Nash, UK; K. J.J. Zwaginga, The Netherlands
Guest Co-Chairs: T. Diacovo, M. Frojmovic

Summary
Speakers covered studies under flow conditions from molecular mechanics to blood coagulation:  bond mechanics of platelet capture (T. Diacovo), platelet amplification loops (M. Hoylaerts), platelet aggregation pharmacology (M. Fromovic), platelet-assisted neutrophil capture from whole blood (G. Nash), monocyte-platelet aggregate adhesion to endothelium (J.J. Zwaginga), and neutrophil-enhancement of platelet procoagulant activity (S. Diamond).

G. Nash
Co-deposition of platelets and leukocytes from flowing blood onto collagen
The study of sequential rolling/adhesion of cells from whole blood to collagen surface is motivated by observations of neurophil rolling on spread platelet lawns under purified conditions.  Citrated whole blood (about 50 uM Ca) supplemented with 5 mM MgCl2 allows integrin function but may also mute platelet responsive and selectin function. Heparin is avoided since it can cause platelet aggregation and antagonism of selectin pathways.  Collagen is most potent for platelet capture and activation when used in a fibrillar form.  Use of fibrillar collagen (100 ug/ml incubation to coat substrate) allows for platelet capture from whole blood, platelet activation, and transient pausing (20-500 msec) of neutrophils, likely through Pselectin-PSGL1 binding.  Use of a downward step flow chamber to set up a recirculation zone (downstream wall shear rate of 450 1/s) provided for marked platelet adhesion in the recirculation zone (max. wall shear rate of –140 1/s) as well as for transient and arrested neutrophil interactions.  The ability of rolling neutrophils to deliver microparticles or other molecules to adherent platelets can be studied in this geometry.  Also the ability of platelet release products to activate adherent neutrophils is significant.  The use of PPACK/Hirudin and/or corn trypsin inhibitor can allow for whole blood flow studies without coagulation at normal calcium.

M. Hoylaerts
Platelet P2X1 in shear stress induced platelet activation.
Platelet release of ADP/ATP presents important pathways for amplification loops of activation.  Platelets have nucleoside receptors (P1 for adenosine) and nucleotides receptors (P2 for ADP, ATP).  They are important drug targets.  Platelets have about 1000 ADP receptors.  P2X1 receptor is an ATP-gated, ADP-antagonized calcium channel with extremely rapid kinetics (msec) compared to P2Y1 and P2Y12 signaling (sec).  Importantly, P2X1 requires calcium for activity.  The receptor is not active under citrated conditions.  An important reagent to allow study of P2X1 is a-b-me-ATP.   The down-regulatory action of ADP on P2X1 provides for many complex effects.  Prevailing flow dynamics and ecto-ATPases (endothelial and potentially platelet) will rapidly control local ADP and ATP concentrations.  The role of mass transfer dynamics and/or mechanotransduction on platelet P2X1 function may play a role in shear-induced platelet activation at pathologic shear rates (about 9000 1/s).


S. L. Diamond
Neutrophil activation of platelets: A role for platelet-derived tissue factor?
In vitro flow systems allow systematic study of neutrophil participation in coagulation.  Adherent neutrophils are highly procoagulant during plasma perfusion is the contact system is active via cathepsin G, elastase, and Mac1-dependent mechanisms.  Use of corn trypsin inhibitor to block XIIa eliminates this activity and allows the study of neutrophil effects on platelet procoagulant activity.   Neutrophil release of cathepsin G can make platelets highly procoagulant via platelet activation by cathepsin G and to a lesser extent Factor X activation by cathepsin G.  The release of cathepsin G by neutrophils may mask any procoagulant action of tissue factor, either neutrophil or platelet derived.   Exposure of convulxin-activated platelets (pretreated with PPACK/GGACK to inhibit VIIa, IIa, and Xa) to purified VIIa, X, and II allow the study of platelet Xase activity which was attenuated by anti-TF and anti-VIIa. TF and other possible VIIa cofactors on activated platelets are detectable under these conditions but their fundamental origin (plasma microparticles, neutrophils, platelets) was unknown.

Tom Diacovo
Similar alteration in bond kinetics define two genetically distinct bleeding disorders: platelet type vs. type 2B vWD.
Presentation of recombinant wt or type 2B A1 domain at exceedingly dilute conditions allows the probing of essentially single bond dynamics with native platelet GPIb or platelet type (PT) vWD platelet GPIb (G233V).   Kinetic analysis revealed that the 2B A1 – wt GPIb and wt A1 – PT GPIb both had similarly slow zero-stress off rates (about 1 s^-1) compared to wt A1 – wt GPIb zero-stress off rate (about 5 s^-1).  The mutants bonds provided for higher tethering frequencies indicating that the on-rate of the mutant interactions may be somewhat higher than the wt on-rate.  These kinetics provide a mechanism for clearance of high molecular weight vWF in 2B or PT vWD.  Translocation velocities of platelets on higher concentrations of wt A1 or 2B A1 coated surfaces are consistent with the bond off-rates since slow off-rates were predictive of slow translocation velocities.  Single bond kinetic analysis provides a fundamental molecular mechanical underpinning to adhesion studies under flow.

Mony Frojmovic
Biorheology of action of antithrombotic drugs
The ability of platelets to stably aggregate can be dependent of prevailing shear rate, receptor activity, pharmacological antagonism, temperature, and donor-specific issues.   PRP prepared with 2 U/ml hirudin/40 uM PPACK allows for study of platelet aggregation in a concentric cylinder viscometer at normal calcium and plasma millieu without fibrin production.  The concept that the IC50 of a pharmacological agent can be dependent on prevailing flow and ambient temperature (22 or 37C) was supported by studies with a P2Y12 antagonist.  At 37C, the IC50 to block platelet aggregation by 50 % increased as the shear rate was increased from 50 to 2500 s-1 for ADP stimulation of aggregation.  Stronger activation of platelets with TRAP required a higher IC50 but was not shear rate dependent.  Protocols are feasible to measure IC50 as a function of shear rate.

Jaap Jan Zwaginga
Monocyte-platelet complexes and their adhesion to endothelium
Platelets are routinely found on monocyte preparations.  Activation of monocytes either in vivo or during isolation protocols will result in co-isolation of platelets and monocyte-platelet aggregates.  Presentation of Lselectin, E-selectin ligand 1 (ESL1) and PSGL1 by monocytes allows for interaction with endothelial Pselectin, Eselectin, and CD34 mucins presented by activated endothelium.  Additionally Mac1, LFA1, and VLA4 can maintain firm arrest via endothelial ICAM1, ICAM2, and VCAM.  The ability of platelet presentation of Pselectin on monocyte-platelet complexes helps to capture these complexes to TNFa-activated HUVEC under flow.  These deposited complexes often form string like features, further highlighting the role of fluid mechanics in their deposition.

Suggestions from the audience
Future meetings should consider examining the state of the art of existing and desirable flow systems, predictive of pathophysioogical and pharmacological  behaviour of blood-vessel wall ineractions  in animal models and in human patients.

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.  A special focus on vWF and its interaction with ristocetin and botrocetin may be particularly relevant to diagnostics for vWD.
  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. A new chair (Dr. J.J. Zwaginga) will be assisted by new co-chair (Dr. T. Diacovo) as well as consultants such as Eric Gabrowski, Mony Frojmovic.