<p>Increased platelet aggregation and Telease of granule contents in response to ADP,. epinephrine, and collagen, increased platelet factor 3-availability induced by ADP or collagen, increased platelet turnover and shortened template bleeding times in patients with type II- and type IV-hyperlipidemia have been reported from several laboratories. Furthermore, studies in experimental animals and in man have indicated that diet-induced elevation of blood Iipid levels may be associated with increased platelet adhesiveness, increased platelet factor 3-availability in response to ADP, and increased platelet turnover. Recently, evidence has been reported that in vitro exposure of pIatelets to increased levels of cholesterol in the medium may be associated with an increase in platelet cholesterol/phospholipid ratio and platelet aggregability. It has also been demonstrated that changes in phospholipid-fatty acid composition of the platelets may occur as a result of diet-induced changes in phospholipid-fatty acid composition of the plasma. Although long chain saturated fatty acids are known to induce or enhance platelet aggregation in vitro, uptake of free fatty acids appears to occur only when the concentration of free fatty acids exceeds the plasma albumin free fatty acid binding capacity. Phospholipids constitute a major proportion of the platelet membranes. Phosphatidic acid and the phosphoinositides appear to be involved in platelet shape change, aggregation, and the platelet release reaction. Certain phospholipids in platelets, when exposed, are powerful accelerators of blood coagulation reactions in vitro.</p> <p>The primary aims of this study were to investigate a) the possibility of a direct exchange of phospholipids between plasma and platelets in vitro; and b) the possibility that phospholipid exchange could result in changes in platelet function.</p> <p>In preliminary studies evidence was obtained to indicate that sodium pentobarbital used as anesthetizing agent in the blood collection procedure in rabbits inhibited platelet adherence to collagen-coated surfaces and the platelet release reaction when the compound was added to platelets in vitro. This effect of SPB appeared not to be related to SPB-induced suppression of platelet energy production but rather due to platelet membrane stabilization. The inhibitory effect of SPB on platelet function was readily reversible and SPB-induced anesthesia in rabbits was not found to cause measurable inhibition of pIatelet function.</p> <p>A sensitive and practical assay for collagen-induced platelet factor 3-availability; using a modified Stypven-clotting time method, was elaborated. During these methodological studies, new evidence with respect to the mechanism of platelet factor 3-availability was obtained. The findings indicated that with platelets from rabbits, pigs and humans platelet factor 3 did not become available in association with platelet shape change or platelet aggregation, but was rather closely linked to the platelet release reaction. However, a small amount of lysis was consistently observed in association with the platelet release reaction and it could not be clearly differentiated whether increased platelet factor 3 availability was truly a result of fusion of granule membranes with the external plate let membrane during the release reaction or a result of release-associated platelet damage.</p> <p>When hyperlipidemia was induced in rabbits by feeding them a diet enriched with egg yolk for a minimum period of 16 weeks, platelets isolated from the blood of these rabbits and resuspended in Tyrode-albumin solution showed a significant increase in collagen- or thrombin-induced aggregation and serotonin release, but not in aggregation induced by ADP. Collagen-induced platelet factor 3-availability was also greater with platelets from hyperlipidemic rabbits compared with platelets from control rabbits. There were no significant differences between the diet and control groups in platelet aggregation induced by ADP, collagen, or thrombin or serotonin release induced by collagen or thrombin in citrated platelet rich plasma. With washed platelets, there was no correlation between the percentage increase in aggregation induced by collagen or thrombin and the plasma cholesterol or triglyceride levels in hyperIipidemic or control rabbits. When washed normal rabbit platelets were resuspended in citrated plasma from a hyperlipidemic rabbit, their response to aggregating or release-inducing stimuli was not significantly different from that observed when platelets from normal rabbits were resuspended in normal plasma. When washed normal were incubated for 30 minutes in hyperlipidemic plasma at 37°C, isolated from the plasma and resuspended in Tyrode-albumin solution, their response to aggregating or release-inducing stimuli was not significantly different from that observed with normal platelets incubated in normal plasma. These findings, thus, support previous reports that diet-induced hyperlipidemia in rabbits may induce an intrinsic functional alteration of the platelets which cannot be readily reproduced by short-term in vitro exposure of platelets to hyperlipidemic plasma.</p> <p>The possibility of a direct exchange of preformed phospholipids between pIatelets and plasma was explored by adaptation of a model previously used in the study of phospholipid exchange in erythrocytes. Washed rabbit platelets were resuspended in plasma in which all of the major phospholipids had been isotopically labeled by injection of ³²PO₄ into rabbits. At certain intervals during a 6-hour incubation at 37°C, aliquots were removed from the incubation mixture and the platelets were isolated and subjected to lipid extraction and phosphilipid analysis. A continuous rise in platelet non-lipid-bound and lipid-bound radioactivity was observed-throughout the incubation period. Two platelet phospholipids, lecithin and lysolecithin, were significantly labeled, whereas little or no labeling of the other phospholipids was found. There was no detectable change in total or individual platelet phospholipid content. At 6 hours, 4% of total platelet phospholipids, 43% of platelet lysolecithin, and 7% of platelet lecithin were labeled. Platelets incubated in plasma from rabbits with diet-induced hyperlipidemia took up and incorporated significantly more label into their phospholipids than did platelets in normal plasma but the pattern of labeling (PC + LPC) was similar to that observed normal plasma. Labeling of both platelet lysolecithin and lecithin could be explained by uptake and metabolism of plasma lysolecithin by platelets. However, labeling of platelet lecithin could at least in part be the result of direct exchange of this phospholipid with the plasma. This is the first demonstration that platelets take up and metabolize endogenous plasma lysolecithin and, possibly, directly exchange lecithin and that these processes may be significantly accelerrated in hyperlipidemic plasma. Uptake and incorporation of endogenous plasma lysolecithin by platelets and, possibly, direct exchange of platelet lecithin appear to be important for platelet membrane phospholipid renewal and could be an important mechanism by which plasma lipids could modify platelet membrane phospholipid-fatty acid composition and, possibly, platelet function.</p> <p>In view of the possibility that lysolecithin might be an important link in the interaction between platelets and plasma lipo-proteins and apparent contradictions in the data reported from other laboratories, the effects of lysolecithin on aggregation, serotonin release, shape and lysis of rabbit, pig, or human platelets in platelet-rich plasma or Tyrode-albumin solution were examined during prolonged incubation. Lysolecithin added to citrated or heparinized PRP from humans or rabbits at a final concentration above 100 μM, caused instantaneous inhibition of platelet aggregation induced by ADP, epinephrine (human PRP only), collagen or thrombin. The inhibitory effect of lysolecithin was found to be partially reversible over a period of 60-90 minutes. Lysolecithin at final concentrations above 30 mM also caused inhibition of ADP-, collagen- and thrombin-induced aggregation and collagen and thrombin-induced release of serotonin in suspensions of rabbit, pig, or human platelets. With washed platelets, the inhibitory effect not only rapidly disappeared, but was followed by transient potentiation of aggregation and serotonin release. This potentiating effect of lysolecithin was most pronounced when thrombin was used as stimulus. Both inhibition and potentiation were observed at concentrations of lysolecithin that did not cause a significant change in platelet shape or loss from platelets of lactic dehydrogenase. Inhibition and potentiation were also observed when plafelets were added to suspending medium containing lysolecithin, although considerably higher concentrations of lysolecithin were required under these conditions. Potentiation was not observed when lysolecithin was added to citrated or heparinized rabbit or human PRP or to washed rabbit platelets suspended in a medium containing 4% bovine serum albumin. It seems Iikely that some or all of the observed effects of lysolecithin on platelet function are due to structural modification of the platelet membrane insufficient to result in gross membrane damage of platelet lysis. In addition, the results of experiments using ¹⁴C-Iysolecithin seemed to indicate that the reversal of inhibition and the potentiating effect of lysolecithin on platelet function may at least in part be related to its rapid uptake and metabolism by the platelets. Whether plasma lysolecithin plays a role in the regulation of platelet function under normal conditions and/or in hyperlipidemia and whether acute changes in plasma lysolecithin concentration (e.g. in response to heparin administration) can acutely modify platelet function remains speculative.</p> <p>Lysophosphatatidylethanolamine and Iysophosphatidylserine also caused inhibition of platelet aggregation and serotonin release in suspensions of washed rabbit and human platelets induced by ADP, collagen or thrombin. With washed pig platelets lysophosphatidylethanolamine inhibited platelet aggregation and the platelet release reaction whereas lysophosphatidylserine induced platelet aggregation and serotonin release. All of these effects on platelets were observed at concentrations of the lysophospholipids which did not cause gross platelet membrane damage or platelet lysis. The differences in the effects of these lysophospholipids on platelet function could be related to marked differences in their fatty acid composition.</p> <p>The results of this study contribute to our knowledge of the mechanisms of platelet factor 3-availability, platelet membrane phospholipid renewal and the role of plasma lipids in the modification of platelet phospholipid and phospholipid-fatty acid composition and platelet function.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/7886 |
| Date | 03 1900 |
| Creators | Joist, Heinrich J. |
| Contributors | Mustard, J.F., Medical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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