Con respecto al objetivo específico “1”

T h e i m p o r t a n c e of p l a t e l e t a d h e s i o n and hy p e r a g g r e g a b i l i t y in the p a t h ophysiology of vascular d i s o r d e r s has led to the widespread use of a n t i - p l a t e l e t drugs in cerebral, myocardial or peripheral ischaemia. The m o s t well e s t a b l i s h e d and commonly used anti-platelet agent is A S A (Fuster et al., 1993). This drug suppresses platelet ag g r e g a t i o n by irreversibly acetylating c y l o o x y g e n a s e hence inhibiting A A metabolism and prev e n t i n g TXAg synthesis (Blackwell et al., 1977; Dahl and Uotila, 1984; G r e s e l e et a l . , 1991).

A n u m b e r of large trials examining the e f fect of a n t i ­ pl a t e l e t drugs on the prevention of further cardiova s c u l a r events (secondary prevention trials) have been c a r ried out in the a b ove patient groups (Anti-platelet Trialists' Collaboration, 1988; Fuster et al., 1993). These studies have d e m o n s t r a t e d that in survivors of myocardial infarction or stroke and in patients with unstable angina or transient ischaemic attacks, A S A is an effective prev e n t i v e a g e n t of r e ­ infarction.

The benefit from ASA intake in the inhibition of a t h e r o g e n e s i s has not been so favourable and remains co n t r o v e r s i a l (Peto et al., 1988). Clearly, by inhibiting c y c l o o x y g e n a s e activity, ASA blocks the formation not only of plat e l e t acti v a t i n g eicosanoids such as PGGg, PGHg and TXAg but pr o b a b l y a lso that of the platelet inhibitor, P G I 2 . This may

be important since PGI2 has potential a n t i - a t h e r o g e n i c p r o p e r t i e s (Moncada and Higgs, 1987). Probably the most important reasons for the limited success of A S A t h e r a p y is that only one of the multiple pathways leading to p l a t e l e t activ a t i o n is blocked by ASA. Thus, although e f f e c t i v e at inhibiting aggregation induced by agonists w h i c h m o b i l i z e A A for TXAg synthesis (e.g. collagen) (Best et al., 1980), A S A is less effective at inhibiting aggregation induced by other agonists (e.g. platelet activating factor, 5-HT, and ADP; K u ster and Frohlich, 1986; De Clerck et al., 1985b; L o u d e n et al., 1992). This has led pharmaceutical companies to deve l o p d r ugs or examine existing drugs for a n t i - platelet properties. The possi b i l i t y that 5-HT may play a role in d i s e a s e states assoc i a t e d w i t h abnormal platelet function has also r a i s e d the interest in anti-5-HT drugs as potential a n t i - p l a t e l e t agents

(Vanhoutte, 1990; Barradas and Mikhailidis, 1 9 9 2 a ) .

1.27 Ketanserin and naftidrofuryl

K e t a n s e r i n (KET) is a relatively selective 5- H T 2 recep t o r a n t a g o n i s t w i t h a moderate a^-adrenoceptor a n t a g o n i s t effect (Fonseca et al., 1984). In its initial clinical e v a l u a t i o n KET ap p e a r e d to possess anti-hypertensive p r operties and perhaps, improve symptoms in patients w ith intermittent c l a u d i c a t i o n and Rayna u d ' s phenomenon (Vanhoutte et al., 1988). Studies a s s e s s i n g the effect of KET on platelets in h u mans h a v e shown that K E T inhibits both in vitro and ex vivo p l a t e l e t a g g r e g a t i o n induced by 5-HT (Bevan and Heptinstall, 1983; The P A C K trial group, 1989; Vanags et al., 1992).

Naftidrofuryl (NAF; Praxilene) is a drug p r e s c r i b e d to PVD p atients to improve walking distance (De Felice et al., 1990). This drug has been shown to p o s s e s s anti-5-HT p rope r t i e s using isolated vascular tissue (Zander et al., 1986; V a s h i s h t et al., 1992). High concentr a t i o n s of N A F (50- 100 jLimol/L) were required to inhibit 5-HT induced a g g r egation in PRP (Davies and Steiner, 1988) and one ex vivo study where NAF was infused into healthy volunteers (0.7 mg/Kg) failed to show any effect on 5-HT induced aggre g a t i o n (Davies and Steiner, 1988). A t present, there are no studies exam i n i n g the e ffects of NAF on ex vivo human p latelet a g g r e g a t i o n in PVD patients.

1.28 M i l r i n o n e

As descr i b e d above (section 1.12), cAMP modul a t e s p l a t e l e t a g g regation and release by c o n t r o l l i n g the m o b i l i s a t i o n of calcium via effects on a g o n i s t - i n d u c e d signal t r a n s d u c t i o n mechanisms, PL-C and PLA2 ' M u c h interest has been shown in drugs that elevate or maint a i n i n t r a c ellular cAMP concentrations. Such drugs include the e i c o sanoids (e.g. PGE^, P G I g ) , the P G l 2“analogue, iloprost (Ashby, 1990) and cAMP- p h o s p h o d i e s t e r a s e (PDF) inhibitors (Patelunas et al., 1991). PDF inhibitors prevent the hydrolysis of cAMP to A M P which m a k e s t h e m p o tential inhibitors of p latelet h y p e r a g g r e g a b i l i t y and t h r o m b o s i s (Hall, 1993). Milrinone, a new PDF inhibitor, is a c a r d i o t o n i c drug, which until recen t l y was a d m i n istered to p a t i e n t s in h eart failure. This drug is a c A M P - s pecific p h o s p h o d i e s t e r a s e type-III inhibitor and has been shown to

inhibit various aspects of platelet function including p l a t e l e t aggregation (Lindgren et al., 1990; P a t t i s o n et al., 1990; Patelunas et al. 1991; Ozin et al., 1992; J e r e m y et al., 1993) . The effect of milrinone on 5-HT - i n d u c e d plate l e t a g g r e g a t i o n and PSC has not been studied.

1.29 Aims of this thesis

The aim of this thesis was to i n v e stigate PSC, a g g r e g a t i o n and platelet-derived substances, n a m e l y 5-HT, in p a t i e n t s with PVD so as to identify plate l e t a b n o r m alities that could contribute to the increased incidence of c a r d i o v a s c u l a r events observed in this condition.

To investigate PSC, a new method was d e v e l o p e d that a llows the measurement of this parameter in a s t a n d a r d i s e d and re p r o d u c i b l e manner. This method was applied to the study of p l a t e l e t agonists and antagonists in normal subje c t s and p a t i e n t s with PVD.

A whole blood single platelet c ounting m e t h o d was se l e c t e d to study platelet aggregation in PVD patients. PRP p r e p a r a t i o n was demonstrated to be a s sociated w i t h a loss of pl a t e l e t s w h ich may explain the conflicting resu l t s reported w i t h PRP optical aggregation methods.

Previous studies have shown that 5-HT is r e l e a s e d from p l a t e l e t s and it was suggested that this b i o a m i n e is an important m e d i a t o r of platelet aggregation and thrombosis; an at t e m p t was made to substantiate this hypothesis. P l asma and p l a t e l e t 5-HT concentrations were assessed in p a t i e n t s w ith P V D , d i a b e t e s m e l l i t u s , r e n a l d i s e a s e a n d

h y p e r c h o l e s t e r o l a e m i a and these concentrations were rela t e d to r i s k factors and other intraplatelet substances.

Finally, the effects of various a n t i p latelet drugs and p l a t e l e t agonists on the uptake and release of 5-HT was studied. This was carried out to establish the role p l a y e d by s u c h p r ocesses on plasma and platelet 5-HT concentrations d u r i n g treatment w i t h platelet active drugs and in conditions a s s o c i a t e d with plate l e t activation.