Platelet Membrane Involvement in Blood Coagulation

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1 Bld Cells (1983) 9: Springer-Verlag Platelet Membrane Invlvement in Bld Cagulatin H.C. HEMKER, J.L.M.L. van RIJN, J. ROSING, G. van DIEIJEN, E.M. BEVERS, and R.F.A. ZWAAL ABSTRACT. Intact platelets d nt shw prcagulant phsphlipids n their exterir. These phsphlipids are lcated at the inside leaf f the bilayer membrane. They becme available by (a) disrupture f the platelets (mechanical smtical etc.), (b) by a mechanism specific fr the platelets, that we call the membrane flip-flp. Membrane flip-flp translcates prcagulant phsphlipids (mainly phsphatidylserine) in the intact platelet frm the inside t the utside. Thus the intact platelet becmes prcagulant. The trigger fr the flip-flp mechanism is the simultaneus presence f small amunts f cllagen and thrmbin. The cltting factrs IXa and VIIIa bind t the prcagulant lipids t frm the factr X cnverting enzyme and factr Xa and factr Va t frm prthrmbinase. KEY WORDS : Thrmbcyte -Membrane asymmetry -Prcagulant phsphlipids - Thrmbin - Cllagen COFACTOR DEPENDENT REACTIONS IN THE BLOOD COAGULATION CASCADE, The sequence f physilgical reactins that eventually leads t the frmatin f thrmbin is knwn as the bld cagulatin cascade. Each step in this cascade cnsists f the frmatin f an active prtelytic enzyme (a serine prtease) frm its pf-enzyme. Several reactins in the cascade require the presence f s-called nn-enzymatic cfactrs. These cfactrs are divided in tw classes: a) prtein cfactrs, b) negatively charged surfaces. It is generally knwn that these nn-enzymatic cfactrs greatly enhance the rate f the cltting factr activatin reactins in which they participate. The reactins and their cfactrs are summarized in Table 1. A quantitative study f these reactins has becme pssible a) because the prteins invlved can nw be btained in a pure state in large enugh quantities and Department f Bichemistry, Rijksuniversiteit Limburg, Maastricht, The Netherlands

2 304 H.C. Hemker et al. Table 1. Cfactr dependent reactins in bld cagulatin Enzyme Substrate Prtein cfactr Surface Factr XII. Kallikrein Factr XII. Factr VII. Factr VII. Factr IX. Factr Xu Prekallikrein Factr XII Factr XI Factr IX Factr Xl Factr X2 Factr II I The extrinsic factr X activatin 2 The intrinsic factr X activatin High mlecular weight kiningen tissue factrapprtein factr VIII lactr V ' Negatively charged I wettable surfaces. J sulphatides etc. I Neeatiretv charsed I nhjsphlipids +"Ca2 * &ld platelets). J Table 2. Effect f nn -enzymatic cfactrs n relative rates f prthrmbin activatin Factr Xa 1 Factr Xa+Caz+ 3 Factr Xa+Caz+ +phsphlipid 68 Factr Xa+Ca2+ * factr Va 560 Factr Xa + Ca2 + + phsphlipid + factr Va 20,000 Relative rate f prthrmbin activatin b) because the prducts f the reactins can be determined spectrphtmetrically with high accuracy due t the develpment f the s-called chrmgenic substrates [1]. The effects f factr Ya, Ca2* and phsphlipid vesicles (cntaining the negatively charged phsphatidylserine) n the rate f prthrmbin activatin, are shwn in Table 2. Factr Va and negatively charged phsphlipids stimulate prthrmbin activatin by factr Xa independently and multiply their effects. with the cmplete prthrmbinase cmplex, cnsisting f factr Xa, factr Ya, Ca2+ and phsphlipid, a 20,000-fld rate enhancement is bserved. Similar bservatins are made fr the effect f cfactrs in intrinsic and extrinsic factr X activatin and the reactins f the cntact activatin system. MECHANISM OF ACTION OF NON-ENZYMATIC COFACTORS IN PROTHROMBIN AND FACTOR X ACTIVATION Prthrmbin and intrinsic factr X activatin have many features in cmmn. The substrates in bth reactins (prthrmbin and factr X) are vitamin K dependent prteins. They cntain y-carbxyglutamic acid residues, respnsible fr the ca2 + dependent binding t negatively charged phsphlipid surfaces. The enzymes (factr Xa and factr IXa), are als vitimin K dependent prteins. The prtein cfactrs (factr va and factr viiia)

3 Platelet Membrane Invlvement in Bld Caeulatin 305 Fig. 1. General mdel fr the rthrmbin and factr X activating cmplexes. Phsphlipid bilayers, cntaining negatively charged phsphlipids, prmte the assembly f the enzyme-cfactr-substrate cmplex Table 3. Kinetic parameters f prthrmbin and factr X activatin Prthrmbin activatr K* prthrmbin (pm) Z-.* (Ila'min- t'xa t) Xa, Ca2+ Xa, Ca2+, phsphlipid Xa, Caz+, phsphlipid, Va ,919 Factr X activatr K- factr X (pm) V^u*$.a'min-r'IXa 1) IXa, Caz+ lxa, Ca2 +, phsphlipid lxa, Ca2+, phsphlipid, VIIIa s 500 have n enzymatic activity and have a high affinity fr negative phsphlipid surfaces, althugh they lack /-carbxyglutamic acids. A general mdel fr the prthrmbin and factr X activating cmplexes is shwn in Figure 1. Phsphlipids prmte the assembly f the enzyme-cfactr-substrate cmplex (factr Xa- and factr Va- prthrmbin and factr IXa- factr VIIIafactr X) via the abve described interactins f cltting factrs with the phsphlipid surface. Althugh it is clear that this type f enzyme cmplex functins much mre efficiently than the enzyme withut accessry factrs (Table 2), the rate measurements d nt permit cnclusins abut the mde f actin f the accessry factrs in the erlzymatic mechanism f cltting factr activatin. Therefre we determined the effects f accessry cmpnents n the kinetic parameters f prthrmbin and intrinsic factr X activatin f2, 3]. This enabled a precise quantitatin f earlier bserved rate enhancements and is the frrst step in elucidating the rle f accessry cmpnents in the mechanism f cagulatin factr activatin. The kinetic parameters f prthrmbin and intrinsic factr X activatin, bserved fr enzyme cmplexes f varying cmpsitin, are summarized in Table 3. In the absence f accessry cmpnents, prthrmbin and factr X activatin are

4 306 H.C. Hemker et al Table 4. Effect f phsphlipid n K- f prthrmbin fr prthrmbin activatin and K- ffactr X fr intrinsic factr X activatin Prthrmbin activatin Intrinsic factr X activatin Phsphlipid (pm) K- app (pm) Phsphlipid (pm) K- app (pm) ) In this experiment n factr Va r factr VIII" was added very inefficient prcesses. The v^u* (that is the turnver number f substrate mlecules by the cmpletely saturated enzyme) is very lw and the K- (the substrate cncentratin causing half maximal enzyme saturatin fr prthrmbin and factr X are cnsiderably higher than the respective plasma cncentratins (prthrmbin 2 pm, factr X 0.21t X[). The presence f phsphlipid plus Ca2 * causes, in bth cmplexes, a drastic drp in the K- t values belw the plasma cncentratins, while the prtein cfactrs greatly increase the V^u*. The bserved changes f the kinetic parameters explain the earlier bserved rate enhancements caused by accessry cmpnents. THE ROLE OF PHOSPHOLIPIDS Since this paper deals with platelet membrane (phsphlipid) invlvement we will nt further discuss the effects f the prtein cfactrs, but fcus n the mde f actin f the phsphlipid. Bth in prthrmbin and intrinsic factr X activatin, phsphlipids cause a drastic drp in the K- fr prthrmbin and factr X, respectively. Als the K_ is dependent n the phsphlipid cncentratin present in the reactin mixture (Table 4). When the phsphlipid cncentratin is increased, the K- increases in parallel. S the K-, measured in the presence f phsphlipid, must be an apparant K- and is nt, as such, a reactin cnstant f the activatin under study. A mdel that can explain bth the drp in K^ and the apparent character bserved in the presence f phsphlipid is presented in Figure 2. In free slutin, enzyme (factr Xa r factr IXa) and substrate (prthrmbin r factr X) have a lw affinity fr each ther, which explains the high K- measured under these cnditins. When negatively charged phsphlipids are included in the reactin mixture, bth the enzymes and substrates

5 Platelet Membrane Invlvement in Bld Cagulatin,< pd A,< 'b*agu * ic q 6,t7- t6a-d*.a) Yq?f p, O q 5 Fig. 2. Mde f actin f phsphlipids in prthrmbin and factr X activatin I??????-????????? rb fff,ff rnflfl fffi Rn Bff fflntrtr Rffi lltr h li id surf ace iiiiii\nr( \r)nnr (/ t888sit8888e888usu 8uu8J uuijiug enzyme(x",lx")? substrate(pt,x)? prduct(lla,xa) bind t the phsphlipid surface via calcium bridges between their 7-carbxyglutamic acid residues and negatively charged head grups f phsphlipid mlecules. This makes a reactin pssible between phsphlipid bund enzyme and phsphlipid bund substrate and thus favurs the number f cllisins between enzyme and substrate. This is the same effect as an increase in cncentratin fthe substrate wuld have. Because the real cncentratin in the reactin vessel des nt change by the additin f phsphlipid the cncentratin f substrate at the phsphlipid surface expresses itself as a drp in K-. Half maximal saturatin f enzyme with substrate, in the presence f phsphlipid, is reached at a much lwer substrate cncentratin because it is determined by the prthrmbin cncentratin at the phsphlipid surface. T attain the same prthrmbin density at the phsphlipid surface at higher phsphlipid cncentratins, mre prthrmbin is required, which explains the bserved increase in the apparent K^ at increasing phsphlipid cncentratins. In this mdel, it is expected that althugh the apparent K* increases with the phsphlipid cncentratin, a K- expressed in terms f cncentratin f phsphlipid bund substrate,

6 308 H.C. Hemker et al. a I E Fig. 3A', B. Effect f phsphlipid cncentratin n the apparent K- and the rkexpressed in substrate surface density fr prthrmbin (A) and intrinsic factr X activatin (B) c O' E Y :l E t00 PHOSPHOLtptD (rrm) a E G Y E x D< trl =l < = wuld be cnstant. 9ri"q binding parameters (dissciatin cnstant (K) and cncentratin f_binding sites) fr prthrmbin and factr x bindin; t phsphlipid vesicles reprted by Nelsestuen [4], we calculated the cnl centratin f substrate (prthrmbin and factr X) bund per pm phsphlipid, at_ the K-'s presented in Table 4. Figure 3 shws the meaiur"d' K- fr prthrmbin and factr X activatin and the amunt f prthrmbin and factr X bund per pm phsphlipid ar varying phsphiipid-cncentratins. Indeed the K- expressed in terms f phspfilipid-bund substrate is independent f the phsphlipid cncentratin, whicl supprts but des nt prve the prpsed mdel. _- N9t all phsphlipids functin equally well in prthrmbin and factr X activatin. In prcagulant membranes net negaiively charged phsphlipids have t be present; natural phsphlipids are inictive.butthermre a liquid crystalline state f the membrane is required fr maximal prcagulant activity [5].

7 Platelet Membrane Invlvement in Bld Caeulatin 309 Table 5. Phsphlipid requirement f prthrmbinase Vesicle cmpsitin (mle %) Prthrmbin activatin PS PI PC PE Sph Chlesterl ('/-) (nm lla'min-r) ' s Intact latelets Platelel phsphlipid extract Platelet snicate z5 1,168 1,176 PS Phsphatidylserine, Pl Phsphatidylinsitl, PC Phsphatidylchline, PZ'Phsphatidylethanlamhe, Sph Sphingmyelin Vesicle and platelet phsphlipid cncentratin is 1 pm, prthrmbin 4 pm, factr Va 30 nm, factr Xa 15 nm THE ROLE OF PLATELETS IN PROTHROMBIN AND FACTOR X ACTIVATION In viv platelets prvide the prcagulant surface required fr cntact activatin reactins as well as fr prthrmbin and intrinsic factr X activatin [6, 7]. The prcagulant activity f platelets in prthrmbin and factr X activatin is knwn as platelet factr 3. Intact nn-stimulated platelets are hwever inactive in cltting factr activatin (Table 5). Fr cmparisn, rates f prthrmbin activatin are given, btained with vesicles cmpsed f the phsphlipids present in the platelet membrane. When the net negatively charged phsphlipids (phsphatidylserine and phsphatidylinsitl) are mitted frm the vesicles in which the phsphlipid cmpsitin f the platelet plasma membrane is mimicked, a lw rate f prthrmbin activatin is measured. In cntrast, artificial vesicles with a cmplete phsphlipid cmpsitin as fund in platelets and als vesicles made frm a cmplete platelet phsphlipid extract, give equally high rates f prthrmbin activatin. Table 5 therefre shws that the net negatively charged phsphatidylserine is essential in prducing full prthrmbin activatin. Phsphatidylserine is hwever almst exclusively lcated in the inner mnlayer f the platelet membrane [8]. It can fr instance be expsed t cltting factrs in the reactin mixture if the platelets are lysed by ultrasnicatin (Table 5). Lysed platelets exhibit the same prthrmbin activatin'-rzftes as vesicles cmpsed f platelet phsphlipids. This suggests that nly phsphatidylserine cntributes t the enhancement f prthrmbin activatin by lysed platelets. When intact platelets are stimulated with the physilgical platelet triggers, cllagen r thrmbin, at cncentratins which give maximal platelet aggregatin and sertnin release, small rate enhancements are measured in assay systems fr prthrmbin and intrinsic factr X activatin

8 310 H.C. Hemker et al. Table 6. Effect f platelets in prthrmbin and intrinsic factr X activatin Platelet stimulated by: Prthrmbin activatin (nm lla'min 1) Factr X activatin (nm X"'min 1) Thrmbin (1.3 nm) Cllagen (10 g/ml) Thrmbin (1.3 nm) plus cllagen (10 g/ml) Cncentratins: )Z L.J pm prthrmbin, 15nM Xa,30nM Va fr prthrmbin activatin 0.5 pm X, 150 nm IXa. 15 nm VIIIa fr factr X activatin Table 7. Sites fr prthrmbin and factr X activatin n human platelet membranes Platelet stimulatr Thrmbin Thrmbin * cllagen Prthrmbin activatin (sites/platelet)? 50n 3,000 26,000 Factr X activatin (sites/platelet) 900 1,200 19,000 (Table 6). The increase f bth activities can be explained by the small amunt f platelet lysis brught abut by the admixture f the reagents. Thrmbin plus cllagen will nt cause mre lysis than each reagent alne. When cmpared with unstimulated platelets, platelets triggered by the cmbined actin f cllagen plus thrmbin hwever shw a tenfld increase f activity in prthrmbin activatin, and a 20-fld rate enhancement in factr X activatin. Bevers et al. [8] fund that platelets, which were triggered by the cmbined actin f cllagen plus thrmbin, expsed their internally Tcalized phsphatidylserine t the membrane exterir. This intrductin f phsphatidylserine in the uter mnlayer f the platelet membrane prduces a platelet surface which prmtes the assembly f bth the prthrmbin and intrinsic factr X activating cmplex by binding f vitamin K dependent cltting factrs. This explains the bserved rate enhancement f thrmbin plus cllagen stimulated platelets in prthrmbin and factr X activatin (Table 6). Experiments as presented in Table 6 can be used t quantitate the number f prthrmbin and factr X activating cmplexes n the platelet surface. In rder t d this, the cagulatin factrs were added in saturating amunts s as t ccupy all functinal sites n the platelets surface and mrever t measure under Z-u* cnditins. Z-u* values btained with phsphlipid vesicles as a prcagulant surface (2,700 min fr prthrmbin and 500 min fr factr X activatin) are used in these calculatins. The amunts f calculated sites (Table 7) fr unstimulated and thrmbin activated platelets are relativelv lw as cmpared with

9 Platelet Membrane Invlvement in Bld Cagulatin Jll Fig. 4. Time curse f appearance f platelet activity in prthrmbin and factr X activatin. Cagulatin factr cncentratins : fr prthrmbin activatin, 4 pm prthrmbin, 15 nm factr Xa, 30 nm Factr Va, fr factr X activatin, 0.5 pm factr X, 50 nm factr IXa and 0.1 nm factr VIIIa. 100% activity in prthrmbin activatin is 800 nm'min- I. IIa frmed, l00/ activity in factr X activatin is 17 nm'min- 1 factr Xa frmed. Platelets were stimulated with thrmbin plus cllasen F1 J x E q U z u tu ^ tactr x activatin. prthrmbin activatin r MINUTES AFTER PLATELET ACTIVATION platelets stimulated by cllagen plus thrmbin tgether. It is nt clear whether unstimulated platelets, under physilgical circumstances, indeed have this lw but signifrcant amunt f functinal binding sites r that they are partially activated. Als a part f this activity must be attributed t the inevitable lysis that ccurs during platelet islatin prcedures. Fr the cllagen plus thrmbin stimulated platelet, apprximately 26,000 functinal binding sites fr the prthrmbinase cmplex and apprximately 20,000 fr the factr X activating cmplex have been calculated. These numbers are s clse t each ther that they strngly suggesthat at saturating cagulatin factr cncentratins, the same sites functin in prthrmbin and factr X activatin at the surface f stimulated platelets. This bservatin suggests that the essential cmpnent fr bth the binding f the prteins f the prthrmbin and the intrinsic factr X activating cmplexes is the phsphatidylserine, that after platelet triggering becmes expsed in the uter mnlayer f the membrane. The time curse f the appearance f prcagulant activity in stimulated platelets can be fllwed in the assay systems fr prthrmbin and intrinsic factr X activatin. In Figure 4 the generatin f a prcagulant surface is fllwed at saturating factr X4 Ya and prthrmbin cncentratins in the prthrmbin activating assay, and at sub-saturating factr IXa and VIIIa cncentratins in the factr X activating system. Half maximal factr X cnverting activity is bserved within 2 min after additin f cllagen plus thrmbin, whereas prthrmbin cnverting activity reaches the half maximal value after 9 min. The bserved differences between the time curses seem t cntradict the idea that the essential cmpnent f the prcagulant surface f stimulated platelets is exclusively phsphatidylserine. This difference can be explained hwever withut pstulating ther prcagulant cmpnents. The requirement f prcagulant phsphlipid as a cmpnent f the

10 3t2 H.C. Hemker et al. be 60 ' Fig. 5. Effect f phsphlipase A, n the activity f cllagen plus thrmbin stimulated platelets in prthrmbin and factr X activatin. Cagulatin factr cncentratins were as described in Fig.4. Stimulated platelets were either treated (clsed symbls) r nt (pen symbls) with phsphlipase Ar. The triangles represent prthrmbin activatin, the circles factr X activatin Incubatin time with PL -ase A2 (min) functinal sites fr the prthrmbin and factr X activating cmplexes culd be determined in phsphlipase digestin experiments. Platelets are stimulated with cllagen plus thrmbin until maximal prcagulant activity is reached and subsequently treated with Naja naja phsphlipase 42, an enzyme knwn t degrade phsphlipid mlecules expsed at the uter platelet surface [7]. Using high phsphlipase A, cncentratins, bth prthrmbin and intrinsic factr X cnverting activities f the stimulated platelets are cmpletely ablished within 10 min. The absence f leakage f lactate dehydrgenase frm the platelets during incubatin with the phsphlipase, indicates that the platelet membrane remains intact. S the cnclusin can be drawn that prcagulant phsphlipids are an essential cmpnent in the functinal sites fr prthrmbin and factr X activatin n the stimulated platelet. When small amunts f phsphlipase A2 are used, the time curse f decay f prthrmbin and factr X cnverting activity f the stimulated platelets culd be fllwed. After different time intervals f phsphlipase treatment, the cagulatin factrs are added t measure the remaining prcagulant activity. Figure 5 shws that the factr X cnverting activity f platelets is mre sensitive t phsphlipase treatment than the platelet prthrmbin cnverting activity. It was investigated whether the differences in time curse f generatin f a prcagulant platelet surface and the sensitivity t phsphlipase A, treatment, as estimated with prthrmbin and factr X activatin, culd be related t different phsphlipid requirements. Therefre, prthrmbin and factr X activatin are measured in mdel systems, using phsphlipid vesicles with varying phsphlipid cmpsitin. Prthrmbin and factr X cnverting activities are measured, using the same cagulatin factr cncentratins as in the experiments with platelets. The vesicles are cmpsed f lipids at the same mlar cncentratin as fund in platelet membranes, with the exceptin f phsphatidylserine and phsphatidylchline. The mle fractin phsphatidylserine is varied

11 Platelet Membrane Invlvement in Bld Cagulatin JIJ Fig. 6. The effect f phsphlipid vesicles with i varying mle percentage : phsphatidylserine in H prthrmbin (clsed symbls) G and factr X activatin (pen.e symbls).100% activity in E prthrmbin activatin is 2,900 ; nm.min 1,1000 activity rn Be factr X activatin is 71.5 nm' mln ^ phsphatidylserine ( m le% ) at the expense f phsphatidylchline and the verall phsphlipid cncentratin is kept at 2 pm. The rates, given as percentage f the maximal activity, measuled fr prthrmbin and factr X activatin at varying mle percentage phsphatidylserine, are given in Figure 6. Larye differences in phsphatidylserine fequirement fr prthrmbin activatin and factr X ictivatin are bserved. Fr the intrinsic factr X activatin, a mle fractin f 20h phsphatidylserine is required fr maximal activity, whereas fr prthrmbin activatin 2.5/ phsphatidylserine is ptimal. At a surface cntaining less than 2.5% phsphatidylserine, factr X activatin rates are negligible, while prthrmbin activatin still prceeds at a cnsiderable rate. This phenmenn prvides the explanatin fr the bserved differences in phsphlipase sensitivity f stimulated platelets in prthrmbin and factr X activatin. If phsphatidylserine in the uter membrane is degraded by phsphlipase Ar, such that the mle fractin phsphatidylserine in the membrane becmes less than 2.5A, still a cnsiderable rate f prthrmbin activatin can be measured, whereas factr X activatin is almst cmpletely ablished. Platelets stimulated with cllagen plus thrmbin expse 25/ f theit phsphatidylserine at the platelet uter surface [8], it can be calculated that the verall mle fractin phsphatidylserine in the uter mnlayer f the platelet is at mst 5%. A mdel phsphlipid vesicle cntaining 5 mle % phsphatidylserine wuld shw a rati f the rates f prthrmbin activatin and factr X activatin f 200: 1. Platelets stimulated ft 2 min with thrmbin plus cllagen give a rate lati f 15 : 1. S the phsphatidylserine expsed at the uter surface f the cllagen plus thrmbin stimulated platelet, cannt be randmly distributed in the uter mnlayer f the platelet membrane. Dmains with higher mle pelcentage phsphatidylserine must exist in the uter mnlayer f the platelet membrane after stimulatin with cllagen plus thrmbin. Otherwise the bserved rati f prthrmbin and factr X activatin rates cannt be explained. Mst f these phsphatidylserine enriched dmains are frmed within 3 min after platelet stimulatin, as can be cncluded frm the time curse f generating factr X

12 I 314 H.C. Hemker et al. I Ctt"s"n / Thrm bin Fig. 7. Mdel fr the generatin f a platelet prcagulant surface. Phsphatidylserine mlecules are indicated by clsed dts cnverting activity. After 3 min platelet stimulatin, mst f the prcagulant surface fr prthrmbin activatin has still t be made (Fig. a). Based n these bservatins, we prpse that the phsphatidylserine, expsed at the surface f cllagen plus thrmbin stimulated platelets, is nt hmgeneusly distributed in the uter mnlayer f the platelet membrane. Dmains with high phsphatidylserine density, which rapidly appear at the surface f stimulated platelets, are measured predminantly with the factr X activating system and t a minr extend with the prthrmbin activating system. Dmains with lw phsphatidylserine, which appear slwer, can nly be measured by the prthrmbin activating system. If these lateilal phase separatins in the uter mnlayer f the membrane ccur during platelet activatin, ther cmpnents may be required t induce r maintain them. In Figure 7a mdel is prpsed, in which after triggering with crlagen plus thrmbin the first phsphatidylserine mlecules that appear at the platelet surface, are clustered by membrane prteins. The prgressive transbilayer mvement after prlnged activatin saturates the clustering prteins with phsphatidylserine. Phsphatidylserine mlecules appearing thereafter are diluted ver the platelet membrane, s frming a dmain with lw phsphatidylserine density favurable fr factr II activatin. REFERENCES 1. flqurn.g, H.9..:,Artificial substrates in Bld cagulatin and Fibrimlyszs. Nijhff Scientific Publishers, The Hague !srNc, J., TlNs, G., Gwi.s-RTEMSLAG, J.W.p., Zwl^tr, R.F.A., Her,rren, H.C..: J. Bil. Chem. 225, , VaN DrnrlnN, G., TaNs, G., RsrNc, J., Hnuren, H.C.:./. Bil. Chem Nur,sBsnEN, G.L., Bnpanrus, M.: Bichemistry 16, , lg77 5.TaNs, G., v.ir.n ZurrunN, H., Cltmr_rnrus, P., Hnurrn, H.C., Zwttr, R.F.A.: Ezr. J. Bichem. 95, , W,trsn, P.N.: Br. J. Haematl l9i2 7. W,l.rsu, P.N.:,Br. J. Haemarl.40, Bavnns, E.M., Cnrrunrus, P., VeN RlrN, J.L.M.L., HrMrnR, H.C., Zwttr. R.F.A.: Eur. J. Bichem. 122,429436, 1982

13 Discussin Discussin 315 OBRINK: Yu have shwn that there is an increase in phsphatidylserine n the utside f the platelets during activatin. D yu knw if that is a functin f flip-flp r if it is insertin f new membrane int the plasma membrane? It culd be membrane vesicles inside the cells which fuse with the plasma membrane. HEMKER: We think it's flip-flp, because fr the phsphatidylserine that appears utside, phsphatidylchline disappears at the same time. That's ne thing. The ther thing is that it can, f curse, be explained by diffusin f vesicles with the uter surface f the platelet. But as far as I've been tld, the asymmetry f the vesicles in the platelets is just the ther way rund frm the asymmetry f the membrane f the platelet itself. S if this is the uter membrane and this is the vesicle, then the phsphatidylserine will be at the inside f the uter membrane and at the utside f the vesicle. S upn fusin, yu will have the same situatin as if there were n flip-flp. This f curse is what we als frnd, because we dn't hnd phsphatidylserine at the utside if we trigger the platelets with thrmbin, in which case there is fusin. FROJMOVIC: Tw small pints. One is that Neville CRawlnl has been publishing papers n snicatin f platelets t essentially try t fractinate membranes, and they hypthese that they may be separating surface cnnecting system frm the surface itself, and that is shwing very distinct bichemical prperties. I just leave this t yu as a pssible thught, that instead f flip-flp, perhaps yu culd have exchange frm the surface cnnecting system t the membrane which is being induced. Can yu rule that ut? Part B, is there a flp-flip? HEMKER: I'm nt quite sure that I gt the gist f yur first questin. As far as we knw, we have n reasn t assume that the surface cnnecting system has a different phsphlipid cmpsitin than the verall membrane. Secndly, indeed there is flp-flip. CAEN: Yu mentined n effect whatsever when using indmethacin. Have yu tried the cllagen-thrmbin interactin with sme cmpund which may affect calcium and even phsphatidic acid? HEMKER: N, we didn't try. NURDEN: Let me see if I remember crrectly. Bth Ken MaNN and Dr Mlrsnus were prpsing that there was a prtease activity in platelets that may be respnsible fr at least sme transfrmatin f Factr V int Factr V-a, and it was hypthesized this might be the calcium-dependent prtease. Nw, ne pssible explanatin fr the defrcit f glycprtein I and V and

14 Jl H.C. Hemker et al. the 17,000 glycprtein in these platelets is that there is an abnrmal, at least fcal, prtease activity in Bernard-Sulier megakarycytes r platelets. It culd be this prtease. It wuld therefre be very interesting t lk at the Factr V in the platelets and its release frm Bernard-Sulier platelets t see if there's any abnrmality there. HEMKER: That was precisely the reasn why yesterday I asked if yu knw anything abut the release f Factr V in Bernard-Sulier platelets. SOULIER: May I ask yu smething, Dr. HsN{KEn? In 1975 Wnrsrr said that in Bernard-Sulier syndrme there was n cllagen activatin at the platelet level. Perhaps cllagen and thrmbin are inadequate when there is a lack GPI. But he als said that there was n Factr XI at all n the platelet, and when mentining ther prteins, yu didn't make any allusin t that. What is yur pinin abut that? HEMKER: In fact, we have been ding these experiments with purified cagulatin factrs, and we dn't have any purified Factr XI arund, s that's the reasn I didn't study it. MILTON: Presumably the reasn why an asymmetry f lipids exists in the membrane is that there is an energy barrier there that stps the lipids ging acrss t ther side. I want t knw if that energy barrier is a functin f the cnfrmatin f the membrane. I'm just wndering if yu cmpletely change the cnfrmatin f the membrane, it may be preferable fr the membrane lipids t cme back ver, just frm the energy pint f view. HEMKER: A membrane that is symmetrical is a mre likely ne than ne that is asymmetrical. I keep asking membrane bichemists, what is the surce f the asymmetry? I get all kinds f answers, but nt very strict nes up t this mment. It might well be, it's very prbable indeed, that there is an interactin between the inside f the membrane and the prteins that are described just lying against the inside f the membrane. We see all kinds f reactins in activated platelets, and amng them might be this interactin between internal prteins and membrane disruptin, fllwed specifically by phsphatidylserine externalizatin. But n the ther hand, that wuldn't explain why it's a reversible prcess. WHITE: In that respect, certainly there are changes in the submembrane area and submembrane prteins. But the interesting aspect f that is in fact, they're quite reversible. We've dne a number f studies n activating platelets usually by aggregating them with ADP and taking apart the aggregated platelets with smething like prstacyclin, and then, thse platelets cmpletely recver their shape, but they're refractry. As sn as yu dissciate the platelets, the phsphrylatin will disappear, and if yu activate them again, it'll redevelp alng with the nw tritn resistant cres, and

15 Discussin 317 then I wuld suspect that the cycling f thse prteins and prtein events will crrelate with the flip-flp r flp-flip f the membrane. HEMKER: I think that's a distinct pssibility, and I wuld just like t suggest that if yu d this type f experiment again, yu shuld nt nly try cllagen and thrmbin but als a cmbinatin f the tw, and whenever yu cme acrss situatins in which the cmbinatin f the tw des smething which is abslutely different frm ne f the tw alne, then yu are very prbably in the regin where the phenmena yu bserve have smething t d with the flip-flp.