Altered Sedimentation Behavior and Ultrastructure of Platelets in Hyperlipidemia

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1 Altered Sedimenttion Behvior nd Ultrstructure of Pltelets in Hyperlipidemi By JEAN W. SALEH, M.D. AND SAMI A. HASHIM, M.D. SUMMARY The influence of hyperlipidemi on pltelet sedimenttion behvior nd ultrstructure ws studied in smples of plsm obtined from 22 norml subjects, 7 ptients with Type II nd 12 ptients with Type IV hyperlipoproteinemi, ll fsting; lso from 11 norml subjects prior to nd t intervls up to 6 hours following ingestion of mel contining 65 g of ft. Pltelet counts were mde on mixed pltelet rich plsm fter centrifugtion of whole blood t 1,000 rpm, on top plsm lyers fter high speed centrifugtion (10,000 rpm), nd on the sediments resuspended in imidzole buffer. The men ± SE number of pltelets per mm3 in top lyer mong norml, Type II nd Type IV plsms were 4,090 ± 405; 9,000 ± 70, nd 21,500 ± 1,950, respectively. The rise nd subsequent fll in plsm triglycerides in response to ft ingestion in norml subjects prlleled those of pltelets in the top lyers. No such correltion ws found with plsm cholesterol. The presence of pltelets in the top lyers of plsm ws demonstrted further by electron microscopy. The buoynt pltelets reveled the presence of osmophilic (lipid) prticles within their open cnliculr system, nd pseudopod formtion. The sedimented pltelets from norml subjects hd smooth contour, wheres those from ptients with hyperlipidemi displyed striking formtion of pseudopods. The dt suggest tht the sedimenttion behvior of pltelets nd their norml structure re ltered significntly s they interct with bnorml concentrtions of plsm lipoproteins. Additionl Indexing Words: Hyperlipoproteinemi Postprndil hyperchylomicronemi Pltelet electron microscopy Hypertriglyceridemi Pltelet buoyncy Hypercholesteremi Pltelet-lipid interction Downloded from by on September 27, 2018 SINCE 1886, blood pltelets hve been known to be essentil for thrombus formtion nd for their bility to dhere to ech other s well s to vessel wlls.' In 1936, Tocntins nd Cntrow2 observed moderte "lipemi" in response to dministrtion of nti-pltelet serum. It ws shown tht plsm freed from ft by high speed centrifugtion could no longer be clotted by ddition of Russel's viper venom, but tht dded ft restored its cogulbility.3 In studies in mn, thrombocytopeni followed repeted infusions of intrvenous ft,4 nd in similr experiment, significnt fll in circulting blood pltelets ws observed fter orl nd intrvenous dministrtion of ft in emulsion form.5 Administrtion of heprin intrvenously three hours fter the ingestion of the emulsified ft showed reduction From the Deprtment of Medicine, St. Luke's Hospitl Center, nd Institute of Humn Nutrition, Columbi University College of Physicins nd Surgeons, New York, New York. Supported by USPHS Grnt AM This work ws done while Dr. Sleh held the Normn Jolliffe Fellowship, Columbi University. Address for reprints: Dr. Smi A. Hshim, Deprtment of Medicine, St. Luke's Hospitl Center, Amsterdm Avenue nd 114th Street, New York, New York Received Mrch 11, 1974; revision ccepted for publiction July 1, in the turbidity of the plsm together with prompt but trnsient rise in the count of circulting pltelets.5 Furthermore, pltelet-rich lipemic plsm is clered less rpidly nd completely thn lipemic but plteletpoor plsm, obtined respectively fter centrifugtion t 1500 rpm nd 3200 rpm.6 Even though it seems cler tht heprin, pltelets, nd ft trnsport nd clering re intimtely relted, no stisfctory explntion of the mechnism of interction of pltelets nd plsm lipids hs been forthcoming. The interction of pltelets with endogenously produced lipoproteins lso hs been subject of interest. Incresed pltelet dhesiveness in hyperlipidemi hs been reported,7' 8 while isolted rbbit nd humn bet-lipoproteins hve been shown to ccelerte significntly denosine diphosphte nd thrombin-induced pltelet ggregtion.9 10 Furthermore, ultrcentrifugtion"' 12 nd electrophoresis" re well known techniques for seprtion of plsm lipoproteins wheres low speed centrifugtion, t 1500 rpm or lower, is used to recover the pltelet-rich plsm (PRP).`4 At higher speeds of plsm centrifugtion, ll pltelets re believed to sediment. Thus, lipids hve been known to interct with pltelets nd to influence their function. The present study ws undertken to determine the sedimenttion behvior of pltelets in plsm from norml subjects Circutltion, Volume 50, November 1974

2 PLATELET SEDIMENTATION/HYPERLIPIDEMIA Downloded from by on September 27, 2018 prior to nd t intervls following ft mel, nd from ptients with Type II nd Type IV hyperlipoproteinemi. Electron microscopic studies were mde of pltelets with ltered sedimenttion behvior. Methods Venous blood smples were drwn in EDTA-contining siliconized glsswre during the post-bsorptive stte from twenty-two norml subjects (8 men nd 14 women, ged 19-71, medin 35), seven ptients with Type 11 (4 women nd 3 men, ged 38-70, medin 60), nd twelve ptients with Type IV (9 men nd 3 women, ged 30-75, medin 50) hyperlipoproteinemi. Phenotyping of hyperlipoproteinemi ws mde ccording to the criteri described by Fredrickson et l.` Type II disese ws documented by the demonstrtion of hevy bet-bnd in the lipoprotein electrophoretogrm, distinct elevtion of plsm totl cholesterol level, norml plsm triglyceride concentrtion, nd cler plsm. Type IV disese ws chrcterized by the demonstrtion of hevy pre-bet bnd by plsm electrophoresis, striking elevtion of plsm triglyceride nd to lesser degree of totl cholesterol concentrtions in lctescent plsm. All blood smples were centrifuged t 1000 rpm for 5 min to obtin pltelet-rich plsm (PRP), nd t 10,000 rpm ("-high speed") for 10 min in Sorvll SS-4 centrifuge. Pltelet counts were mde ccording to the method of Miescher nd Gerrde`6 on mixed PRP nd, fter high speed centrifugtion on top lyers nd on the sediment, the ltter resuspended to originl volume in imidzole buffer (0.05M) contining 1% EDTA. For the purpose of pltelet counting nd electrophoresis, the top lyer of plsm obtined fter "high speed" centrifugtion ws smpled by the cpillry ction of stndrdized twenty microliter disposble pipette brought crefully in contct with the surfce of the plsm. For this purpose, the descent of the pipette to the surfce of the plsm ws controlled by ttching it to stnd equipped with verticl slide. Lipoprotein electrophoresis,17 nd totl cholesterol`8 nd triglyceride19 concentrtions were performed on ll fsting plsms. The effect of ultrcentrifugtion on pltelet sedimenttion ws studied in plsm smples from two norml subjects, two ptients with Type II nd two ptients with Type IV hyperlipoproteinemi. The smples were subjected to centrifugtion t 42,000 rpm for 30 minutes in Beckmn preprtory ultrcentrifuge (Model L2-65B), using TI 50 rotor. In order to chrcterize further the top lyer of plsm fter high speed centrifugtion, electrophoresis ws done on top lyers of plsm from two norml subjects, two ptients with Type II nd three ptients with Type IV hyperlipoproteinemi. In order to study the effect of diet-induced hyperchylomicronemi on pltelet sedimenttion behvior, eleven norml volunteers were fed, fter 12-hour fst, mel contining pproximtely 65 g of ft (three mediumsized eggs, 7 pts of butter, 25 g of bcon, nd 120 ml of whole milk). Plsm smples were obtined prior to brekfst nd t hourly intervls up to 6 hours. Pltelet counts were mde on ech mixed PRP nd, fter centrifugtion t 10,000 rpm, on ech top lyer nd on the sediment resuspended in buffer. Also, plsm cholesterol nd triglycerides were mesured concurrently in ll fsting nd postprndil plsm smples. Electrophoresis ws performed on top lyers of the plsm smples following high speed centrifugtion. The ultimte identifiction of the pltelets in the top Circultion, Volume 50, November 1974 lyers of plsm ws mde by trnsmission electron microscopy. Fsting smples of PRP from norml subjects nd ptients with Types II nd IV hyperlipoproteinemi were fixed in 0.1% glutrldehyde in White's solution20 nd centrifuged t 10,000 rpm. The top lyers of plsm hd n dditionl fixtion in 3% glutrldehyde followed by finl fixtion in 1% osmic cid. The pltelets were trpped on Millipore filter, wshed in buffer, dehydrted in lcohol nd embedded in Epon 812. Thin sections were cut with dimond knife nd stined with led citrte nd urnyl cette. All observtions were mde in Zeiss 9S microscope. The sedimented pltelets of fsting PRP from norml subjects nd ptients with Types II nd IV hyperlipoproteinemi were fixed in similr mnner nd exmined by scnning electron microscopy. For this purpose, the fixed pltelets were trpped on Millipore filter nd coted with suspension of colloidl gold. Scnning ws performed by use of n AMR 900 microscopy set t 20 Kv nd 200 tilt. Results The totl number of pltelets in mixed PRP nd in top lyer of plsm of the norml subjects nd the ptients with Types II nd IV hyperlipoproteinemi, together with vlues of plsm triglycerides nd totl cholesterol re shown in tble 1. In the ptients with Type IV disese, significntly higher proportion of pltelets ws recovered in the top lyer of plsm, when compred with the norml subjects (P <.0001). This lyer, when subjected to lipoprotein electrophoresis in three subjects displyed pre-bet lipoprotein pttern of migrtion. In contrst, in the ptients with Type II disese significntly smller proportion of pltelets ws recovered in the top lyer of plsm when compred with tht from ptients with Type IV (P <.001). In two ptients with Type II disorder, the electrophoretogrm of the top lyer disclosed bet-lipoprotein pttern of migrtion. However, the proportion of pltelets in top lyer of plsm obtined from ptients with Type II disese ws significntly higher (P <.001) thn tht in the top lyer from the norml subjects. It is pprent tht certin proportion of pltelets "flot" in the top lyer of norml plsm, tht this number increses significntly in ptients with Type II disese nd tht such n increse is even higher in ptients with Type IV disese. In contrst, the number of sedimented pltelets derived from ptients with Types II nd IV ws significntly lower (P <.001) thn tht of the norml controls. Also, significntly more pltelets sedimented (P <.001) in the plsms of ptients with Type II thn in those with Type IV disese (tble 1). Ultrcentrifugtion t 42,000 rpm for 30 minutes done on the plsm of two norml subjects disclosed pltelet counts of 410,000 nd 380,000 fter the sedimented "plugs" were resuspended in buffer to the originl volume of plsm, nd the counts in the top plsm lyers were 2,000 nd 3,000. The mixed PRP counts were 412,000 nd 415,000, respectively. Elec-

3 882 SALEH, HASHIM Tble 1 Pltelet Counts, Plsm Triglyceride, nd Totl Plsm Cholesterol Concentrtions Plsm Pltelets in sedimented lipoprotein plugs resuspended to sttuls Top lyer Plsm Plsm totl originl plsm (number of PRP Top lyer "7c of PRP triglycerides cholesterol volumes subjects) (men :1 SE) (men 4 SE) (men * SE) (men 4 SE) (men 4- SE) (men 45 SE) Norml (22) 332,000 13,000 4,090( i f ,000 13,595 Type 11 (7) 288,000 3,600 9, ,000-2,114 Type IV (12) 287,000-6,000 21,00 f1, , Downloded from by on September 27, 2018 trophoresis of the cler top lyers showed extremely fint bet bnds. In the two ptients with Type II hyperlipoproteinemi the counts of the sedimented pltelets were 373,000 nd 335,000; those of the top plsm lyers were 6,000 nd 5,000, while those of the mixed PRPs were 390,000 nd 340,000, respectively. Electrophoresis of the cloudy top lyers displyed hevy bet bnds of the low density lipoproteins. In the two ptients with Type IV disese in whom the pltelet counts in the mixed PRPs were 280,000 nd 390,000, the counts in the top plsm lyers were 15,000 nd 12,000 nd those in the sediments were 260,000 nd 380,000, respectively. Electrophoresis of the "'milky" top lyers disclosed typicl pre-bet bnd migrtion of the very low density lipoproteins. The results of pltelet studies in the eleven norml volunteers fed 65 g of ft s brekfst fter n overnight fst re shown in tble 2 nd figure 1, together with concurrently determined plsm triglyceride nd totl cholesterol concentrtions. The curve representing men serum triglyceride chnges in response to ft ingestion in these norml subjects prlleled tht of men pltelet counts in the top lyers. When compred with the men postbsorptive vlue, the men pltelet counts in the top lyers of plsm t two to five hours following the ft lod rose significntly (P = <.001-<.005). Concurrently, the number of sedimented pltelets decresed, reching significnce (P <.05) t two nd three hours. The men plsm totl cholesterol vlues, clculted t the sme intervls s those for plsm triglycerides nd pltelet counts, did not chnge significntly fter the ft lod. All fsting plsms were cler. Turbidity ws seen t two nd up to five hours fter brekfst. During this time intervl nd following high speed centrifugtion (10,000), the plsms ppered cremy t the top, cloudy t the middle nd cler t the bottom. This distinct seprtion vnished slowly fter the fourth hour following the ft ingestion nd in eight of the volunteers, the plsms gin were cler throughout the tube t the end of the experiment. Electrophoresis of the top lyers crried on on ll the ptients t the pek of hyperlipemi disclosed nonmigrting pttern with hevy concentrtion t the origin, suggesting the presence exclusively of chylomicrons. The plsm triglyceride concentrtions in the postbsorptive stte in the 22 norml subjects, the seven ptients with Type II nd twelve ptients with Type IV hyperlipoproteinemi displyed significnt degree of correltion with the pltelet counts in top lyers of their respective plsms (fig. 2). In contrst, there ws no significnt correltion in the sme subjects between plsm totl cholesterol nd pltelet counts in top lyers of plsm (fig. 3). Tble 2 Pltelet Counts, Plsm Cholesterol nd Triglyceride 65 g of Ft Concentrtions in 11 Norml Adults Follotwing Ingestion of Pltelets in sedimented Top lyer Plsm Plsm totl plugs resuspended to PRP Top ly-er % of PRP triglycerides cholesterol originl plsm volume Houirs (men i SE) (men i SE) (men 4- SE) (men i SE) (men ± SE) (men * SE) 0) 385,909-13,017 4, i ,000 12, ,5,545-12,596 7,300 1, i ,000-12, ,636-11,891 11,400-1, ,000 11, ,363-12,614 13,400-2, ,000 12, ,0 10,379 11,100-2,428 2.X , , 382,727 10,008 9,200-1, ) ,000 9, ,750-3,145 3, ,000 2,886 Circutltion Volume 50, November 1974

4 PLATELET SEDIMENTATION/HYPERLIPIDEMIA ,000- L4ji -_ 150 2,000 E - k ib 1, tj.q) 0 J 15 1~ 5 R c 200 v., ' *v NORMAL 100 v - TYPE II v 80 *-TYPE IW 25x * * * y r 76'25 60 * - n.41 "8 r PLATELETS IN TOP LAYER PERCENT OF PLATELETS IN PLATELET-RICH PLASMA Figure 2 Semi-log plot showing significnt degree of correltion between 6 ttriglyceride concentrtions nd pltelet counts in top lyers of 6 plsm fter high speed centrifugtion. The subjects involved in these correltions were 22 normls, seven with Type II nd twelve with Type IV hyperlipoproteinemi. Downloded from by on September 27, 2018 E_ qk t FAT LOAD 2 Figure 1 HOURS Chnges (men ± SE) in plsm triglyceride () nd cholesterol (c) concentrtions nd pltelet counts in top plsm lyers (b) fter high speed centrifugtion in eleven norml young dults prior to nd t intervls up to six hours following the ingestion by ech subject of 65 g of ft. The pltelets in the top lyers of plsm were identified clerly by trnsmission electron microscopy. The buoynt pltelets from norml subjects (fig. 4) nd ptients with Type II disorder displyed their chrcteristic grnules, few pseudopods nd n open cnliculr system which contined osmophilic mteril indicting the presence of lipid. No osmophilic structures were seen between the pltelets. The floting pltelets from ptients with Type IV disese (fig. 5) displyed similr ultrstructure except Circultion, Volume 50, November for the presence of more lipid inclusions in their open cnliculr system nd more pseudopods; lso, osmophilic structures were scttered outside the pltelets. The sedimented pltelets from norml subjects displyed smooth contour with occsionl excrescences, while those from ptients with Types II nd IV hyperlipoproteinemi disclosed striking for- 6 mtion of pseudopods (fig. 6) , (I) _ E Qt 200- (id r- * 9 5 0~~~~~ y = 9+3x r = PLATELETS IN TOP LAYER PERCENT OF PLATELETS IN PLATELET-RICH PLASMA Figure 3 Semi-log plot showing lck of correltion between plsm totl cholesterol concentrtions nd pltelet counts in top lyers of plsm fter high speed centrifugtion. The subjects involved in these correltions were 22 normls, seven with Type II nd twelve with Type IV hyperlipoproteinemi. *. 0

5 SALEH, HASHIM 884 Discussion The results indicte tht the sedimenttion behvior of pltelets cn be influenced by the stte of the plsm lipids. It is pprent tht certin proportion of pltelets "flots" in norml plsm during high speed centrifugtion. This proportion increses significntly in plsms from ptients with Type II disese. However, in our series the most striking increse in the proportion of floting pltelets ws observed in the plsm from ptients with Type IV hyperlipoproteinemi. Thus, the presence in the plsm of incresed concentrtion of bet-lipoprotein nd to lrger extent of pre-bet lipoprotein ppers to promote "floting" of pltelets. The striking correltion between the top lyer:prp pltelet rtio nd serum triglycerides, nd the lck of correltion between such rtio nd plsm totl cholesterol suggest tht triglyceride-rich lipoproteins re involved in the pltelet "floting" phenomenon. The hyperchylomicronemi induced by orl ft lods in the norml subjects ws ssocited with n increse in the proportion of pltelets in the top lyer of plsm suggesting gin tht hypertriglyceridemi is involved in the flottion of pltelets. It is possible tht the lipoprotein prticles, when present in inordinte concentrtions in the plsm (hyper-bet nd hyperdownloded from by on September 27, i W A SH -' v0f s l pre-bet lipoproteinemis nd hyperchylomicronemi), physiclly dhere to certin type of pltelet thus rendering them buoynt. This possibility lso could explin the occurrence of incresed flottion of pltelets in the plsm from ptients with Type II disese. However, since the degree of pltelet floting in these ptients ws significntly lower thn tht in ptients with Type IV disese, the buoynt effect on the pltelets of the pre-bet-lipoproteins, on the bsis of physicl interction, ppers to be more pronounced thn tht of the bet-lipoproteins. Surfce interctions of pltelets hve been described with vriety of mcromolecules nd moleculr ggregtes including polymerizing fibrin,2' ntigen-ntibody complexes, nd uncoted ltex prticles nd those coted with protein.22 Moreover, isolted humn bet lipoproteins hve been shown to enhnce pltelet ggregtion in the presence of denosine diphosphte or thrombin.`0 The extent to which such in vitro phenomen my be pertinent to modifiction of pltelet sedimenttion by the lipoproteins cnnot be scertined. Another possible mechnism for the ccelerted buoyncy of pltelets derived from ptients with hyperlipoproteinemi my relte to chnged density of the pltelets. A well known physiologic behvior of, 'C b~~~~~~ k Figure 5 Figure 4 Pltelets from top lyer of ultrcentrifuged plsm from norml subject during postbsorptive stte. Pltelets disply their chrcteristic dense bodies, grnules, nd open cnliculr system. Three offour pltelets re disc shped. There re only two sections of pseudopods. Note osmophilic mteril (rrow) in cnliculr system. No osmophilic round structures in bckground. (X 7,200.) Pltelets from top lyer of ultrcentrifuged plsm from ptient with Type IV hyperlipoproteinemi duiring postbsorptive stte. Pltelets disply their chrcteristic dense bodies, grnules, nd open cnliculr system. No disc shped pltelets re seen. There re severl sections of pseudopods. Note incresed osmophilic mteril (rrow) in cnliculr system. Numerous osmophilic round structures re seen in bckground. (X 7,200.) Circtltion, Volume ;50, November 1974

6 PLATELET SEDIMENTATION/HYPERLIPIDEM IA 885 Figure 6 t/i 1pe I1 (c) d(enrieds1 fron i iiornil subject (). ptient iil/i lp II (1) ic ptieiit fitl crogiphi c froni? those sedimienting e/nc hilpe/1r/ipopioz0teii1(11z1(1. 7I/lic pltelets wcrn c r c eiitrifugtion t 10,000rp 1ipii j/nliiitg fitio(nil it/h g r/ittildhliidei. Ii2 co 1iit Is Cc) i/it c pltelef ts fivin (le tnorn1ill sn b(jec t ilhosefronrithl c czt iciis wit f/11prhl pi/c ni i cisplit st.ik inig fori iiiitoii o f hii Scnning efl l1 {lecltrnl i pj.seific/oc/l.s. X of p/e l itels 3, Downloded from by on September 27, 2018 pltelets is their bility to phgocytize vriety of prticulte mteril,22' 23 including emulsions of lipid.24 Also, it hs been shown tht the pltelets in nimls rendered hyperlipidemic by dietry mens undergo chnge in the ftty cid pttern of their lipids in ccordnce with the nture of the dietry ftty cids. For exmple, when butter ws the mjor component of dietry ft, lrger proportion of sturted ftty cids ws observed in the pltelets.25 A similrly incresed proportion of sturted ftty cids ws demonstrted in pltelets derived from ptients suffering from coronry hert disese.25 Thus, it my be ssumed tht t lest some of the ft entering the pltelets is derived from the plsm lipoproteins or the chylomicrons. As shown in this study, the ultrstructure of the buoynt pltelets from norml subjects nd ptients with hyperlipoproteinemi revels the presence of osmophilic mteril, presumbly lipid, within the pltelet open cnliculr system. The presence of these inclusions in the floting pltelets my explin, in prt, the mechnism nd degree of pltelet buoyncy. It is noteworthy tht the buoynt pltelets from norml subjects nd ptients with Type II hyperlipidemi displyed slight degree of pseudopod formtion, while those from ptients with Type IV disorder disclosed n enhnced formtion of pseudopods. Studies hve shown tht formtion of pseudopods by pltelets my be mnifesttion of (:,ircutltion, V/unoe 50, Nouenmber their response to vriety of stimuli.26 It is possible tht the uptke of lipid by the pltelets my induce such structurl response. This mechnism lso my explin pseudopod formtion in the sedimented pltelets from ptients with hyperlipidemi. However, other fctors thn lipid uptke by the pltelets must ply role in their buoyncy, since the mjority sediment. One such fctor is chnge in the density of the plsm consequent to n incresed concentrtion of lipoproteins. Also, the non-lipid components of the pltelets my differ in the buoynt nd the sedimented pltelets. Further investigtion is needed to scertin the extent to which hyperlipidemi is ssocited with chnge in pltelet behvior, in terms of incresed ggregtion or interction with the rteril endothelium. Acknowledgment We re indebted to Dr. Hrry Crter nd Mr. Mrkus Meyerhofer, Merck Institute, Rhwy, N.J. for invluble help in the electron microscopic studies, nd to Mr Sudhir Vdher for technicl ssistnce. References 1. Emis Eiitii CJ, S( ms.nikcf:,in.usc.ii C: Experimentelle untersuchungen uber thrombose. Virchows Arch [Pthol Ant] 130: 39, Tm-ANiINs L, CATAoisno A: The plsm lipids in purpur produced with ntipltelet serum. J Immunol 30: 261, Mx(FuALANE RG, TnEVxAN JW, Ari-iooD AMP. Prticiption

7 Downloded from by on September 27, of ft soluble substnce in cogultion of blood. J Physiol 99: 7P, JOHNSON WA, FREEMAN S, MEYER KA: Some effects of intrvenous emulsions on humn subjects. J Lb Clin Med 39: 176, COLE JW, BIilTT AG, LAULGH1Y CW, HOLDEN WD: The effect of limentry nd intrvenously induced hyperlipemi on pltelets. Surg Forum 4: 173, MI FCHELL JRA: Inhibition of heprin clering by pltelets. Lncet 2: 169, PHILIP RB, WRIGHT HP: Effect of denosine on pltelet dhesiveness in fsting nd lipemic bloods. Lncet 2: 208, RENSENBRINK JF, HOLZKNECHT H, BRAU\NSTEINER H: Ezhohte ggregtion der thrombozyten bei essentieler hyperlipemic. Act Hemtol (Bsel) 38: 95, FARBIZEW SKI R, WOROWSKI K: Enhncement of pltelet ggregtion nd dhesiveness by bet-lipoprotein. J Atherosclerosis Res 8: 988, FARBIZEWvSKI R, SKRZYDLEWSKI Z, WOROWX SKI K: The effect of lipoprotein frction on dhesiveness nd ggregtion of blood pltelets. Thromb Dith Hemorrh 21: 89, HAVEL RJ, EDER HA, BRAGDON JH: The distribution nd chemicl composition of ultrcentrifuglly seprted lipoproteins in humn serum. J Clin Invest 84: 1345, FURMIAN RH, HOWXARD RP, LAKSHMI K, NORCIA LN: The serum lipids nd lipoproteins in norml nd hyperlipidemic subjects s determined by preprtive ultrcentrifugtion. Am J Clin Nutr 9: 73, LEES RS, HATCH FT: Shrper seprtion of lipoprotein species by pper electrophoresis in lbumin-contining buffer. J Lb Clin Med 61: 518, WEISS HJ, ALEDORT LM, KOCHWA S: The effect of slicyltes on the hemosttic properties of pltelets in mn. J Clin Invest 47: 2169, 1968 SALEH, HASHIM 15. FREDRICKSON DS, LEVY RI, LEES RS: Ft trnsport in lipoprotein: n integrted pproch to mechnisms nd disorders. N Engl J Med 276: 34, 94, 148, 215, 273, MIESCHER PA, GERARDE HW: A one-step method for counting leukocytes nd pltelets. Am J Clin Pthol 46: 576, NOBLE RP: Electrophoretic seprtion of plsm lipoproteins in grose gel. J Lipid Res 9: 693, BLOCK WD, JARRET KJ JR, LEVINE JB: Use of single color regent to improve the utomted determintion of serum totl cholesterol. In Automtion in Anlyticl Chemistry. New York, 1965, p KESSLER G, LEDERER A: Fluorometric mesurement of triglycerides. In Automtion in Anlyticl Chemistry. New York, 1965, p WHITE JG: Fine structurl ltertions induced in pltelets by denosine diphosphte. Blood 31: 604, NIEXXVIAROWX SKI S, REGOECZI E, STEWXXART GJ, SENYI AF, MUSTARD JF: Pltelet interction with polymerizing fibrin. J Clin Invest 51: 685, MUSTARD JF, GLYNN MF, MISHIZAMIA EE, PACKHAM MA: Pltelet surfce interctions: reltionship to thrombosis nd hemostsis. Fed Proc 26: 106, WEBER E, MORGENSTERN E, PFLEIDERER Th: On the influence of lipid emulsions on blood pltelets nd leukocytes in vitro. In, Pltelets nd the Vessel Wll - Fibrin Deposition, edited by SCHETTLER G. Stuttgrt, Georg Thieme Verlg, 1970, p Hox IG T, GROTTImNI KA: Lipid infusions in mn: Ultrstructurl studies on blood pltelet uptke of ft prticles. Thromb Dith Hemorrh (Stuttg) 29: 240, RENALD S, Kl-RA K, GOULET C, LEMIRE Y, ALLARD C: Reltionship between ftty cid composition of pltelets nd pltelet ggregtion in rt nd mn. Circ Res 26: 553, BAHNHAR[ MI, WALSH RT, ROBINSON JA: A three dimensionl view of pltelet responses to chemicl stimuli. Ann NY Acd Sci 201: 360, 1972 Circultion, Volume 50, November 1974