Identification of the clinical, functional and anatomical factors that in­ fluence plaque stability have important clinical consequences.622 This concept is one

of many that have provided an impetus to determine if thrombogenic risk factors provide a link between CAD and its clinical expression as CHD.

3. The development of generalised atherosclerosis may merely be a con­ tributing factor to the clinical expression of CAD in the form of CHD. The forma­ tion of acute severe stenoses may have different risk factors and mechanisms than those for the development of diffuse atherosclerosis.4,623

4. However, the lipoproteins are also related to moderate CAD lesions. Such lesions may also be predisposed to rupture with subsequent thrombus forma­ tion. This process may occur without additional factors than rapid accumulation of lipid material in a lesion, in conjunction with alteration in local haemodynamic and shear stress forces, being the cause.

5.3 LIPOPROTEINS AND PLATELET FUNCTION 5.3.1 Hyperlipoproteinaemia and platelet function

A considerable body of evidence exists dem onstrating an adverse effect of hyperli- poproteinaem ias on platelet fu n ctio n .65 G iven, the p resen t u n derstanding o f th eir interaction, the alteration in platelet function occurring in the presence of hyperlipo- proteinaem ia could feasibly contribute to atherogenesis and/or its progression. The changes in platelets that are said to occur in association w ith elevated lipids could also conceivably give rise to the clinical symptomatic forms of the disease.65

The concept of circulating lipoproteins being potentially thrombogenic has long been an area o f investigation.66,67,624,625 Numerous in vivo and in vitro methods o f assess­ ing platelet function have predominantly, although not universally, shown increased platelet reactivity in association with hyperlipoproteinaemia.66,67,625 This interaction appears to be particularly consistent for hypercholesterolaemia.

5.3.1.1 Platelet aggregation. A relationship between increased platelet aggreg- ability and dyslipoproteinaem ias has been well estab lish ed .65,66,126,421,423,424,626,627 The available information relating the effect of lipoproteins on platelet sensitivity has been obtained from the investigation of subjects with hyperlipoproteinaem ia, which have in general been supported by studies on isolated platelets.420 There may be a threshold level of LDL to effect platelet aggregation, with low LDL concentrations having little influence on aggregation.420 There is an altered response by platelets to ag g reg atin g agents follow ing p retreatm en t w ith the m ain lip id density fractions (L D L , V LD L and H D L ).420,421 F u rth erm o re, a g reater e ffe c t on in vitro p la te le t aggregation of normal platelets is seen with the lipoprotein fractions from hypercho- le s te ro la e m ic p a tie n ts th an is seen w ith the fr a c tio n s fro m n o rm o lip id a e m ic c o n tro ls.419 O f further interest is the inverse effect o f HD L com pared to LD L and V L D L .419'421 P latelets enriched with cholesterol ex vivo are m ore re a ctiv e 422 and produce more TXB2 on aggregation.126,128,422

In vitro studies have in general supported cross-sectional observational studies. Plate­ let re a c tiv ity may be affe cted by the lip o p ro tein c o n ce n tra tio n w ith in a norm al population.423 Hypercholesterolaemic individuals may have enhanced in vitro platelet reactivity com pared to norm al subjects.124,424,425 Increased ADP sensitivity in Frie- d rick so n ’s Type II hyperlipidaem ic patients could result in platelet aggregation in vivo under conditions in which none would occur in normals. Even more striking is the hypersensitivity of such persons to aggregation by adrenaline. The low concen­ tration o f adrenaline required for in vivo aggregation in patients with Type II hyper- lipidaem ia m ight occur under certain conditions such as severe stress or traum a.424 Such observations have not been universal. In another report, specific in vitro plate­ let aggregation responses to collagen and ADP in hyperlipidem ic patients with CHD did not d iffer significantly from norm als. H ow ever, there appeared to be some in vivo platelet activation in response to ADP and collagen.426 Platelets from patients w ith ty p e II h y p erlip o p ro tein a e m ia are m ore sen sitiv e to ag g reg atio n by ADP, a d re n a lin e and c o lla g e n ;124 th o se from type IV p a tie n ts d id not d iffe r from the norm al control group in their responses to ADP and collagen, but they were more sensitive to adrenaline.124 W ithin a population o f healthy m ales, it has been shown

that total and LDL cholesterol concentrations influence the sensitivity of platelets to adrenaline and less so ADP.420,423 There was no correlation with HDL or VLDL concentrations, as observed by others.67 Platelet hypersensitivity is also induced by cholesterol incorporation into the platelet membrane.126 Others, however, have been unable to find any alteration in the sensitivity of platelets to aggregation by ADP, adrenaline and collagen in patients with type II hyperlipidemia,127 although the dif­ ference may be explained by dissimilarities in laboratory methodology.

5.3.1.2 Cyclooxygenase activity and metabolism. The amount of TXB2 pro­ duced after arachidonic acid-induced aggregation of washed platelets may be closely correlated with lipid levels.505 These observations support the results of another study which examined thrombin induced production of TXB2 in PRP.628 In that study, increased TXB2 production was found in both type II and IV patients, but more so in the type II patients; the amount of TXB2 correlated with the LDL cho­ lesterol level and the apoprotein B concentration.628 Furthermore, TXB2 production in clotting whole blood was increased, as was the rate of synthesis of TXB2, in type II subjects.628 The amount of TXB2 produced also correlated with the plasma apo­ protein B levels in type II subjects.628

5.3.1.3 Platelet release reaction. The release of platelet BTG appears to be in­ creased in patients with hyperlipoproteinaemia and peripheral vascular disease. The total cholesterol level increases with age, as does the LDL cholesterol level within industrialised societies. An increase in plasma BTG levels in old apparently healthy subjects has been reported.423,629 Plasma BTG and PF4 levels have been reported to have a strong positive correlation with age, being more pronounced in older females.630 This sex difference has not been reported by others.423,629 If there is in­ creased platelet reactivity with age, the actual influence of increasing LDL levels, or, alternatively, the presence of asymptomatic atherosclerosis, remains to be deter­ mined.

5.3.1.4 Platelet count ratio. Lowe and his colleagues631 studied a group with type II hyperlipoproteinaemia and showed a significant reduction in the PCR in comparison to an age and sex matched control group. However, individuals with clinical arterial disease were included in this study, introducing a susceptibility bias. 5.3.1.5 Possible nature of the platelet-lipioprotein interaction. The presence of a cholesterol enriched milieu may alter the lipid and phospholipid content of the platelet membrane, and hence its function. Shattil and associates used sonicated mixtures of cholesterol and phospholipid (liposomes) to simulate an abnormal envi­ ronment.126 PRP was incubated with the liposomes causing normal platelets to ac­ quire 39% excess cholesterol with no change in the phospholipid or protein content. This was accompanied by a marked increase in platelet sensitivity to aggregation by adrenaline and ADP, without any effect on the response to thrombin or collagen. When platelets were incubated with cholesterol-poor liposomes, there was a 21% reduction in platelet cholesterol, and a marked reduction in their sensitivity to adre­ naline.126 Incubation with liposomes which caused no change in the cholesterol content, resulted in no alteration in platelet sensitivity to aggregating agents.126 Using similar methods others have studied the influence of cholesterol content on the metabolism of arachidonic acid and found a 23% increase in TXB2 release from

Cholesterol rich platelets, compared to 14% from cholesterol depleted platelets in the presence o f throm bin.128 A rachidonic acid and other m etabolites w ere released in significantly higher proportions from cholesterol-rich platelets. The overall increased aggregatory response was asso ciated w ith an absolute in crease in the am ount o f arachidonic acid released as w ell as an increase in cy clooxygenase pathw ay p ro ­ ducts, with no increase in lipooxygenase pathw ay products. These findings have been confirm ed.422 This latter study also exam ined the effect o f an increased m em ­ brane cholesterol to phospholipid ratio on phospholipid metabolism; enhanced hydro­ lysis of platelet phosphatides in cholesterol-enriched platelets stimulated by thrombin was reported.

It is clear from these and other studies that the lipid com position o f platelets may have an influential effect on the function of platelets in man.65,423,627

5.3.2 Methods

The methods for the platelet function studies and lipid measurements are detailed in Chapter 2.

5.3.3 Results

In the control group there was no c o rrelatio n betw een any m easu res o f p la te let aggregation and blood cholesterol, apoprotein B and triglyceride levels. In the group with CHD, the cholesterol level correlated with the rate o f platelet aggregation with a d re n a lin e (r= 0 .4 2 , p < 0 .0 2 ) an d A D P (r= 0 .2 7 , p < 0 .0 5 ), an d in v e r s e ly w ith LT50% aggregation w ith adrenaline (r= -0.27, p< 0.05). A poprotein B only c o rre­

lated with RADR (r=0.35, p<0.02).

The PCR and the serum TXB2 level did not correlate with any of the lipid measures in the case or control group. The plasm a BTG level correlated inversely with apo­ protein A and HDL cholesterol levels in the control group (Table 7.11, C hapter 7). The MPV correlated inversely with the HDL cholesterol level in the case group, as did the triglyceride level (Table 7.1, Chapter 7).

5.3.4 Discussion

The resu lts do not in d ic a te a strong rela tio n sh ip betw een the lip o p ro tein values m easured and p latelet function in norm al subjects, but do so for in vitro p latelet aggregation in the case group. The associations o bserved are co n sisten t w ith in ­ creased in vitro reactiv ity o f platelets to specific aggregating agents (particularly adrenaline) in patients w ith CHD. A sim ilar relatio n sh ip was not observed in the control population. Previous studies of normal subjects have dem onstrated a relation- sip between platelet aggregation (particularly adrenaline induced aggregation), and increasing cholesterol levels.127,419,422,424,426 A possible explanation for this inconsis­ tency m ay re la te to the lack o f in d iv id u als w ith lip o p ro tein a b n o rm alities in the co n tro l group in th is study. M ost prev io u s stu d ies c o n ce n tra te d on groups w ith abnormal lipoprotein profiles. The case group on the average had lipoprotein m eas­ ures d iffering from the controls (e.g. higher ch o lestero l levels), and in the group with CHD the level o f cholesterol did have a relationship with platelet aggregation,

consistent with previously published results.

5.3.5 Summary

The blood cholesterol level must be included in the m ultivariate analysis o f platelet function and CAD, when appropriate, in the present case-control study because:

1. A potential bias from changes in lipoproteins in the case group due to m odification o f diet following the diagnosis of the disease exists. There may still be an effect present although diluted by this change and hence not detectable with the small numbers studied.

2. There exists a large but unclear body o f evidence implicating a relation­ ship between lipoproteins and abnormal platelet function.

3. There is consistency in the literature and in the present study indicating