Reproducibility of the WB-FPC method

The intra-assay variation for plat e l e t counts at 3 00 x 10^/L (basal counts) (n=10) was 4% and 18% for p l a t e l e t counts at 30 X 10^/L following 10 /xmol/L (n=10) ADP. R e p r o d u c i b i l i t y of SPA was also shown to be good w i t h no significant d i f f erences between the initial W B - F P C and the same m e a s u r e m e n t 2 hr later (Barradas et al., 1992b).

3.5 D I S C U S S I O N

WB - I A depends on platelet aggregates a t t a c h i n g to p l a t i n u m electrodes before an impedance change is registered. "Weak" agonists, however tend to form m a n y small r e v ersible aggregates w h ich never reach the critical size to ensure an

impedance change (Sweeney et al., 1989). Particle counters, on the other hand, measure aggregate formation and because time of sampling can be selected, platelet a g g r egation can be a sse s s e d before platelet clumps disaggregate. Thus, plat e l e t c oun t i n g techniques are ideally suited to detect the effect of "weak" agonists or phenomena such as SPA.

Previous experience with W B - I A demon s t r a t e d that high conce n t r a t i o n s of agonists are required before d e t e c t a b l e WB- IA is achieved. In addition, adrenaline-induced W B - I A was only o b s e r v e d in IHD patients following the injection of u n f r a c t i o n a t e d heparin, an established stimulator of plat e l e t ag g r e g a t i o n (Greenbaum et al., 1987).

The results presented in this thesis show that W B - I A cannot detect aggregation induced by 5-HT w h e r e a s WB-F P C a g g r e g o m e t r y is able to do this r e p r o d u c i b l y . The inability of W B - I A to detect aggregation to agonists such as PAF and SPA, as w ell as the requirement for high concentrations of other agoni s t s (e.g. collagen) are serious d i s a d v antages (Barradas et a l . , 1 9 9 2 b ) .

As outl i n e d in Chapter 1, 5-HT release from activated pl a t e l e t s has been proposed as an important m e d i a t o r of e n h a n c e d plate l e t and vascular reactivity in conditions a s s o c i a t e d w i t h atherosclerosis. Clearly, in order to assess the role of platelets and associated m e diators in disease states asso c i a t e d with atherosclerosis, p l a t e l e t a g g r egation r e s p o n s e s to such mediators should be a s s e s s e d and i n v e s t i g a t e d w i t h a method with optimum sensitivity. Despite the reser v a t i o n s regarding WB-IA outlined above, this

t e c h n i q u e was used by Elwood and colleagues (1990) to d e m o n s t r a t e a significant association between A D P - i n d u c e d WB- IA a n d IHD. In some circumstances, and w i t h part i c u l a r agonists, W B - I A is clearly a useful method and m a y complement o t h e r t e c hniques to assess platelet function.

Part 2

3.6

Platelet aggregation in patients with PVD

A reas o n for abandoning PRP-based optical a g g r e gometry w a s the w i d e l y held v iew that the c e n trifugation proc e d u r e r e q u i r e d for PRP preparation might be associated w i t h the loss of platelets. Furthermore, in h yperaggregable conditions, this c e n t r i f u g a t i o n may affect platelet function in a varia b l e m a n n e r r e s u l t i n g in enhanced, diminished or u n c h a n g e d plat e l e t aggregation. The effect of spinning on p l a t e l e t y i e l d and f u n c t i o n was, therefore, assessed in PVD p atients and healthy subjects.

3.7 METHODS