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Effect of fibronectin and von willebrand factor on the adhesion of human fixed washed platelets to collagen immobilized beads
THROMBOSIS RESEARCH 44; 661-672, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
EFFECT OF FIBRONECTINANDVON WILLEBRANDFACTORON THE ADHESION OF HUMAN FIXED WASHED PLATELEESTo COLLAGENIMMOBILIZEDBEADS
M. Aihara, H. Takami, Y. Sawada,S. Morimoto,K. Kariya, I. Kudo, K. Ueno, A. Kimura and Y. Yoshida First Departmentof InternalMedicineand Blood Transfusion Service,HirosakiUniversitySchool of Medicine, Hirosaki,Japan (Received 24.2.1986; Accepted in revi'sedform 11.8.1986 by Editor M. Matsuda)
The adhesion of human fixed washed platelets(FWP)to collagen was measured using collagen immobilized beads. The addition of normal plasma or severe von Willebrand disease (VWD) plasma to FWPd ecreased the adhesion, suggesting the presence of some inhibitors of platelet adhesion in human plasma. Although the adhesion of FWP in severe VWD plasma was not different from that of FWP in normal plasma, the additionof purified von Willebrand factor(vWF,l-2 u/ml ristocetin cofactor) to FWP in buffer increasedthe FWP adhesion at higher flow rates, and the percent of adhesion in the absence of vWF was 10 %(collagen 500 ug) and 30% kollagen 1,000 ug) of that in the presenceof vWF at 10 ml/min. The enhancing effect of the vWF on FWP adhesion was also observed by pretreatment of the collagen column with vWF suggesting the important role of bound vWF to the collagen; adhesion 72% to the collagen column(l,600ug) treated with vWF and 16 % to the collagencolumn without the pretreatment at 10 ml/min. The promoting effect of vWF was also present in some commercial factorVII1 preparations which had no large or intermediatemultimers of vWF antigen.The adhesionof FWP was inhibited by fibronectin(FN) and the binding of ristocetin cofactor(vWF:RoO)to collagen fiber was also inhibited by FN; bound vWF:RCoto 50 ug/ml collagenin the absence or presence of 125 ug/ml FN were 60% and 8% respectively. It is suggestedthat vWF, even small multimer of vWF:Ag, is involved in the initial platelet-collagen interactionat high flow rates, while plasma FN acts as one of anti-adhesionfactor. ------___-_-----_
Key words:Human platelets,platelet adhesion,collagen,fibronectinand von Willebrandfactor. Reprintsrequeststo Dr. M Aihara, 5 Zaifucho,Hirosaki036, Japan 661
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1NTR0DuCr1oN Adhesion of blood platelets to the site of vascular injury is one of the first steps in hemostatic plug formation. The reaction is generally considered to be the adhesion of platelets to exposed subendothelial connective tissues(l,2). There are several factors involved in this interaction, such as von Willebrand factor, fibrinogen, factor XIII, fibronectin and thrombospondin(2). The role of the von Willebrand factor(vWF) in platelet adhesion to subendothelium or collagen has been reported using several different technigues(3-5). We have previously reported that von Willebrand factor related antigen (vWF:Ag)increasesthe rate of adhesion of human fixed washed platelets(FWP) to collagen in suspension,and that high molecular weight forms of vWF:Ag preferentially bind to the fibrillarcollagen(6).However, it is still uncertainthat how vWF enhancesplateletadhesion to collagenand what kind of form of vWF is needed for the reaction. which is also high molecular weight Concerningabout fibronectin(FN), glycoproteinand involvedin platelet-subendothelium interaction,there are several different observations about an effect on platelet-collagen interaction. Although the requirement of FN for platelet adhesion to collagen has been described(7,8),inhibitory effect of FN on plateletcollagenhas been also reported by several groups(9,lO). In this paper, we describe a role of vWFand FN on platelet-collagen interaction.The adhesionof FWP to collagenwas inhibitedby normal plasma or plasma of the patient with severe von Willebrand disease(VWD). PurifiedvWF increasedthe adhesionof FWP to the collagenat a higher flow rate and an increased effect of vWF on adhesion was also observed after pretreatmentof the collagenbead column with vWF, suggestingthe important role of bound vWF in mediatingplateletadhesion.Furthermore,the adhesion of FWP to collagen was also increased by several commercial factor VIII preparations,which showed the lack of large or intermediate multimers of vWF:Ag. PurifiedFN, however, did inhibitnot only the adhesion of FWP to collagenbeads but also the bindingof vWF:RcO to collagenin suspension. MATERIALSANDMEX'HOIX
Materials: Reagents were allreagentgrade unless specified. Water was deionizedand glass distilled.Bovine serum albumin(BSA, crystallized and lyophilized) was obtained from Sigma Chemical Co.(StLouis), and human serum albumin(HSA)and human gamma globulin fraction were purchased from Green Cross Co.(Japan). Sepharose CL-4B was from Pharmacia Fine chemicals(Uppsala, Sweden). Agarose and Gel Bond Film for electrophoresis were from FMC Corp., Rockland, ME. sodium dodecyl sulfate(SDS, electrophoresis purity reagents)was from Bio-RadLabs, Richmond,CA. Human FN was purchased from Biomedical Tech., Inc., MA. and Cooper Biomedical Inc.,PA. The buffer used in the adhesionexperimentswas 0.05M caccdylateO.lM NaCl, pH 7.3(cacodylate buffer). Assay of vWF:RCo, vWF:Ag and FN:
The activity of vWF was measured as ristocetin cofactor(vWF:RCo) ssing a Payton dual channel aggregometer(Buffal0, NY). FWP(8 x 10 /ml), 0.2 ml was mixed with 0.2 ml of normal plasma dilutions or test materials in a siliconized cuvette and the mixture was then added by 0.2 ml of 4.5 mg/ml of ristccetin(H.Lundbeck Co., Copenhagen, Denmark). !Ihe initial slope of the aggregation curve was
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measured and used for the guantitation of vWF:RCo(ll).BothvWF:Ag and FN were measured by Laurel1 electroimmunoassay(l2) using 1% Seakem ME agarose and I:200 rabbit anti-human vWF:Ag which was prepared in our laboratoryand rabbit anti-humanFN(Behringwerke AG, Marburg,West Germany). ckleunit of these activitiesis, by convention,that amount found in 1 ml of pooled normal human plasma. Plateletpoor plasma: Blood was collectedwith informedconsentfrom twelve normal adult donors and one patient with severe VWD(vWF:RCo < 0.03 u/ml, vWF:Ag < 0.03u/ml) into1/9volumeof 3.2% sodium citrate. Platelet poor plasmas were centrifuged for 20 min at 3,000 rpm at 4OC, and then recentrifugedat 10,000rpm for 30 min at 4OC before stored at -7OOC CommercialMI1 preparations:CommercialfactorVIII preparationsused were Confact-F(Chemo-Sero-Therapeutic Res., Japan), Haemate-P(Hoechst, Div.; BoehringwerkAG), Hykryo(NipponSeiyaku Co.,.Japan),Hemofil-HT(Hyland Travenol Labs., USA), Conco eight-HT(Green Cross Co., Japan) and Kryobulin(Immuno AG.,.Austria). The ratios of vWF:RCo/vWF:Ag and the concentration of FN(u/ml) were 0.63, 1.9 in Confact-F, 0.59, 7.4 in Haemate-P, 0.49, 23.2 in Hykryo, 0.30, 30.2 in Hemofil-HT, 0.25, 29.1 in Conco eight-HT and 0.25, 8.8 in Kryobulin. For the adhesion experiments, each preparationwas diluted with cacodylatebuffer and the amount of vWF:Ag was adjustedto l-2 u/ml. Purified factor VIII/vWF complex: Human FVIII/vWF complex was purified from commercial FVIII preparation(Hykryo, Kryobulin) as previously described(l3). The commercial preparation was added by 25% polyethylene glycol to a final concentration of 5.5% and mixed for 30 min at 4OC. The precipitatewas collectedby centrifugationfor 30 min at 3,000rpm, at 4Oc and dissolved in 0.05M Tris-0.3MNaCl, 5 mM citrate, 50mM c-amino-caproic acid(EACA),pH 7.3. The material was adjustedat a concentrationof 5 mg/ml and was again precipitatedby polyethyleneglycol. The dissolved material was loaded onto Sepharose CL-4B equilibulated in the buffer and peak fractions in the voidvolume were pooled and dialyzed against cacodylate buffer. The supernatantmaterial after centrifugation 10,000 rpm for 30 min at 4OC was stored at -7OOC. vWF:Aq multimer: The multimeric forms of vWF:Ag were analysedby the method of SDS agarose gel electrophoresis using 1.5% Seakem HGT(P) agarose. The multimers were visualizedby immunoenzymatictechniqueusing avidin-biotin peroxidasecomplex as previouslydescribed(l4).Briefly,the gel was washed with distilledwater after the electrophoresis and reactedwith I:100antivWF:Ag in 1% human gamma globulin solutionovernight. The gel was washed with O.OlM phosphatebuffered saline(PBS)containing0.05% Tween-20, pH 7.4 and then incubatedwith l:lOO-200biotinylatedgoat anti-rabbitIgG. The gel was then incubated for 4hrs with avidin biotin-peroxidasecomplex(Vector labs. Inc.,Burlingame, CA).The peroxidase activity was visulalized with the substrate, 60 mg 4-chloro-I-naphthol(Bio-F&d), 0.06 ml of 30% hydrogen peroxidein 20 ml methanol-100ml PBS. Collagen& collagen coated glass beads: Dispersedbovine Achilles tendon collagen was obtained from Ethicon Inc., Somerville,NJ(a gift from Dr. RL Kronenthal).The material was shown to.bepolymeric.byelectronmicroscopic studies (15). The stock solution of collagen was prepared by adding the original collagen suspension (14 mg/ml) to the appropriate volume of distilled water. The mixture was rocked overnight at 4OC and then stored
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until used. 'Ibprepare collagencoated glass beads, 5ml of the collagen in distilledwater(250-2,000 ug/ml) was neutralizedby the addition of 0.8-1.0 ml of O.lM ammonium bicarbonate in a plastic box. Fifteen grams of glass beads(O.3-0.4 mm diameter, Eiken, Japan) were then added to the mixture and the mixture was vacuum dried overnightat room temperature. Fixed washedplatelets(FWP): FWP were prepared by a modification of the method-lain JP eta1 (16). Human vlatelets concentrates were washed with 6mM titrated saline; pH 7.1 and-were fixed with 1.8% formaldehyde solution, pH 6.4 overnight at 4'C. After fixing, the FWP were washed and then stored at 4OC, 8 x IO8 platelets/ml in 0.05M imidazole buffer containing ImM ethylene diamine tetraacetic acid(EJITA) and 0.02% sodium azide, pH 6.4. For the adhesion experiments, the FWP were resuspended in cacodylatebuffer. (luantitation of the adhesion--of FWP to collagenimmobilizedbeads: Adhesion -of FWP to collagen was tested by a column filtration method which was modified from an early system described by Fauville and Davis(l7). Approximately 3 gm of collagen coated glass beads were packed intoa 4 mm diameter vinyl chloride tube(Kohsin, Japan) under negativepressure. The FWP(1.5-2.0x 108/m1)ina IO mlsyringe were thenapplied to the collagen column at the constant flow rate using an infusionpump(MicroFeeder JP-S, Furue Science, Japan). Each 0.5 ml fractionwas collectedinto the plastic tubing and platelet count was measured by an electroparticle counter(Thrombocounter-C, Coulter, FL). The degree of adhesion was calculatedfrom the differencebetween the count of non adherentFWP in the passage through the column and that of initial FWP solution,and expressed as percent of the initial control. Because the adhesion was decreased by washing the collagencolumn with buffer prior to the applicationof FWP, the columns of dried collagenbeads were used in most experiments. Totestan ef ect of plasma, purifiedvWF, or FN on FWP adhesion to collagen,FWP(4 x IO6/ml) in buffer containingl-2% BSA was mixed with equal volume of test materialsand used for the adhesionexperiment. Morphologicalobservationof the adherent platelets was performed by scanningelectron microscopy. The collagen bds were removed from the column after the adhesionexperimentand prefixedwith 1% glutaraldehydein O.?M phosphate buffer, pH 7.4 overnight. After washing the beads with phosphatebuffer, the samples were post-fixedwith 1% osmic acid(WakoPure Chemical Ind.,Japan) for 2-3 hours and then dehydrated by acetone. The materials finally evaporated with carbon and gold were observed by a scanningelectronmicroscope(Nippon Eenshi, Japan). Binding experimentswith collagen: Normal human plasma, 0.3 ml was mixed with 0.3 ml of the collagen suspension in a siliconized aggregometer cuvette. The mixture was stirred at a setting of 1,000 rpm for IO min at 37OC using a Payton aggregomter. The supernatant, after removal of the collagenaggregatesby centrifugationfor 5 min at 3,000 rpm, were assayed for residualactivitiesof vWF and FN. Binding of vWF to the collagen was also evaluated using purified vWF(4 u/ml vWF:RCo) and the effect of FNon vWF adsorptionto collagenwas tested.After two millilitersof the purified vWF was mixed with 2 ml of 500 ug/ml FN or cacodylatebuffer, 0.3 ml of the mixture was then added by equal volume of collagen in suspension. The supernatant materials after the centrifugation were assayedin a similar manner.
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RESULTS FWP adhered to collagen coated glass beads without platelet aggregation(Fig 1). Most platelets attached to the collagen fibers were unspreadedand their patternswere similar to those of the contactplatelets accordingto the definitionby Baumgartner(18). Adherence--of FWP to collagen coated glass beads: In the control experiment using vinyl chloride tubing and glass beads without collagen, FWP in caccdylatebuffer did not adhere to the tubing, but did adhere to the glass beads. The number of platelets passed through the control glass beads column(30cm) was 2.6% in first fraction,6.8% in second fraction,17.0%in third fraction and 30.0% in forth fraction at the f ow rate of 1 ml/min, when compared to the originalplateletcount(2.0x 10B/ml). This nonspecificadhesionwas partly decreased by either increasing the flow speed or increasingthe size of glass beads in the column(datanot shown here). Bovine serum albumin(BSA) also inhibited the nonspecific binding of FWP to the glass beads without collagen. When FWP resuspendedin BSA at the concentration between 0.5% and 5%, the binding of FWP to the glass beads was completelyabolishedat the concentrationof BSA above 0.5%. Therefore, cacodylate buffer with 0.5% BSA was used in the adhesion experimentswith the collagencoated glass bead column. Effect of ----flow rate on the FWP adhesionto collagen: FWP were applied to theiiagen column(the amount of cXlagen, 1,000ug/column) and each 0.5 ml fractionwas collected. At the flow rate of 1 ml/min, the percent of adhesion in the fraction from first to sixth were 67.9%, 61.7%, 55.5%, 48.8%, 46.0%,and 42.0% respectively(Fig.2). At a higher flow rates such as 5 or 10 ml/min, however, the percent adhesion in each fraction was constant from the first 0.5 ml. To evaluate aneffectof the flow rate or collagenconcentrationon the FWP adhesion,percentplateletadhesionto the collagen column was arbitrarily defined as the mean values of four
FIG. 1 Adhesionof FWP to collagen.FWP were mixed with collagenin suspensionor collagen immobilized beads, and the mixtures were observed by scanning electron microscopy. (A) collagen in suspension(bar 1 urn), (B) collagen coated glass beads(bar5 um).
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FIG. 2 Effect of flow rate on the adhesion of FWP to collagen.Conditions: platelet 1.5-2.0 x log/m1 in caccdylatebuffer with 0.5% BSA; collagen, 1,000 ug/column;fractionsize, 0.5 ml; flow rate, l(A), 5(B), 10(C) ml/min. The degree of adhesion (mean value of first 4 fractions) were 58.5% (A), 24.4%(B) and 13.1%(C) respectively.
FIG. 3
250
500
COLLAGEN
750
1000
CONCENTRATION
”
2000
IpglColumnl
Effectof collagenconcentration on the platelet adhesion. Conditions were the same as in Fig. 2 except that the amount of the collagen in column was varied from 250 to 2,000 ug. The percentadhesion increased with an increasing amount of collagen in the column at all flow rates, but decreased with increasing flow rate at the constant amount of collagen.
fractions(lst-4th,2ml). The effects of varied amounts of collagen were tested at 1, 5 and 10 ml/min flow rates. The percent of plateletadhesion increasedwith an increasingamount of collagen in the column at all flow rates(Fig. 3). With a given amount of collagen in a column, the percent adhesion decreased with increasing flow rate. Percent adhesion to the column containing 500 ug collagen was 40.120.8% at Iml/min, 9.5+ 1.2% at 5ml/min and 3.8+ 0.8%at IO ml/min(mean+ SD, n=3). Effect of vWF on FWP adhesion to collagen : -----
When purified vWF was introducedinto the test system, thYadhesion of FWP to collagencolumn was significantly increased. With the glass bead column containing 1,000 ug collagen,the percent adhesion in the absence of vWF was 58.5%at the flow rate of 1 ml/min, while 76.5% adhesion was observed in the presence of vWF(lable1). The enhancingeffect of vWF on adhesion increasedwith flow rate; adhesion in the absence of vWF was 10.9%(collagen 500 ug) and 30.2%(collagen1,000ug) ofthevalue in the presence of vWF at IO ml/min. The effect of vWF on adhesion was dose dependent and the percent adhesion(500 ug collagen) was 78.5%(vWF:RCo 4u/ml), 64.8%(2 u/ml) and 24.8%(1u/ml) at 5 ml/min. The effect of vWF on FWP adhesion to collagencolumn was also tested by pretreatment of the column with vWF. The collagen coated glass bead column(30cm length, 1,600 ug collagen)was previouslywashed with 5 ml of either buffer or purifiedvWF(vWF:ROo4-5 u/ml) at a constantflow rate of 2
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TABLE I Effect of Purified VWF on FWP Adhesion to Collagenat different Flow Rate(Collagen 1,000 ug/column;final concentration of vWF, I-1.5u/mlvWF':RCo; Value, I&an + SD, n=3, *p
Without
VWF
With
VWF
1
58.5 + 1.3* 76.5 5 1.4 5 24.4 T 1.6* 60.0 2 1.0 10 13.1 z 2.6" 43.2 2 2.0 -__--_--_---_--__--_--~---_--~--~_--~--~~--_--~------~------~---------~-
TABLE II Effectsof mmercial MI1 Preparationson FWP Adhesionto collagen 3 /ml were mixed with test materials,controlbuffer; (FWP 4 x 10 cacodylatebuffer containing1% EEA, Value; mean 2 SD, n=3, ND: not done, P
collagen
500 uglcolumn 2000 ug/column (5 ml/min flow rate) (IO ml/min flow rate) -~--------------------_--~---_--_------_------_---_--_--__--__-_________ controlbuffer 6.0 + 0.5 29.4 + 3.2* confact-F 14.9 T 2.9** 55.5 71.7" Haemate-P 17.0 + 0.6* 61.8 + 0.8" Hykryo 6.7 7 2.3 47.6 7 0.4* Conco eight-HT 10.0 T 0.1* 54.7 T 0.4* Hemofil-HT 6.8 T 0.3 63.8 T 1.7* Kryobulin 5.6 7 0.8 44.6 + 0.3* PurifiedvWP 24.8 z 1.9* GD -_--_------_-_--_-__-~---~~-~----~-~~---~-~------~--~------~------------
ml/min. The solution remaining inside the column was then removed by passing sufficientair at a flow rate of 10 mllmin. The adhesion0 FWP to collagen was then examined by filtering the FWP(1.5-2.0 x 10B /ml in caccdylatebuffer without RSA) through the collagencolumn at flow rates of 0.5, 1, 3, 5 and 10 ml/min. Each 0.5 ml fraction was collected, but the first 5 drops were excluded to minimize the dilution of FWP in the first fraction because of the wet column. The percent adhesion of FWP to the collagen column pretreatedwith vWF was higher than that of the untreated collagen column, especially at high flow rates; the adhesion to the column without pretreatmentwas 93.8% at 0.5 ml/min, 75.7% at 1 ml/min, 54.7% at 3 ml/min, 51.6% at 5 ml/min and 16.0% at 10 ml/min, while the adhesion to the column with pretreatmentwas 92.4%at 0.5 ml/min, 95.0% at 1 mllmin, 94.1% at 3 ml/min, 87.9% at 5 ml/min and 71.9% at 10 ml/min(meanof duplicate).
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Effect of commercial FVIII preparations -on FWPadhesionto collagen: The Be& patterns of six commercialFVIII preparationsw= shown in Fig. 4. All preparationswere characterizedby lack of large multimer of vWF:Ag and furthermore,four except confact-Fand Haemate-P showed the absence of intermediate multimer. The triplet structure of each small multimer was abnormal in all preparations and the fastest migrating subband of the triplet was increased in quantity, while the central band was stained dominantlyin normal plasma. When FWP were added by these preparationsin which vWF:Ag was adjusted to 1 u/ml, the degree of adhesionto collagen(500 ug) at 5 ml/min was significantly increased by Confact-F, Haemate-P and Conco eight-HT,although the enhancingeffects on adhesion were lower than that of purified vWF(Table II). However, when the concentrations of the preparationswere increasedto twice(vWF:Ag2 u/ml), the adhesion measured using 2,000 ug collagen at IO ml/min was increased by all preparations. Effect of plasma -on FWP adhesion to collagen: When FWP(4 x 108/ml) were -mixed with equal volume of normalplasma dilutions, the FWP adhesion to collagendecreasedby increasingthe plasma concentration;the adhesion to collagen column(20 cm, 1,300 ug collagen) was 45.5% in the absence of plasma and 26.0 % (I:2normal plasma), 29.7%(1:4), 44.7%(1:8),46.5%(1:16) and 45.2% (1:32)at 5 ml/min. The inhibitory effect of plasma on FWP adhesionwas also observedby severevWD plasma and the adhesionof FWP in VWD plasma(18.3+1.6%)was not significantlydifferent from that of FWP in normal plasma(21.0-+ 0.8%),even at 5 ml/min flow rate. Effect of FN on FWP adhesionto collagen:When purifiedFN was added to FWP, theadhesionofFWP to collagen column!1300 us) was inhibited dose dependently; the adhesion was 34%(FN, 31 ug/ml), 29%(62 ug/ml), 16%(125 ug/ml), 2%(250 ug/ml) and 51%(intheabsence of ~~)at 5 ml/min flow rate. The inhibitory effect of FN on the adhesion was also observed at 0.5 and 1 ml/min flow rate, and the adhesion was decreasedafter pretreatmentof the collagenby purified F~(datanot shown). Binding of vWF to collagen in suspension: Both FN and vWF:Ag in normal human plasma-a to collagen in suspensionand the dwee of binding was increased by increasing collagen concentration(data not shown). When purified vWF(4 u/mlvWF:RCo) was mixed with 500 ug/ml FN and tested for collagen binding experiment, the residual amount of vWF:RCo in supernatant was higher in the presence of FNthanthat in the absence of FN;the amount of bound vWF:RCo to the collagen(fina1 cont. ug/ml) was 100%(200 and 500 ug/ml), 40%(100 ug/ml), 28%(50 ug/ml)and nil(25 ug/ml) inthe presence of FN; however, 100%(100, 200 and 500 ug/ml), 60%(50 ug/ml)and 32%(25 ug/ml) in the absence of FN. The degree of binding of vWF:RCo in the presence of FN was approximately13% of that in the absence of FN at the concentration of the collagen 50 ug/ml(Fig. 5). This inhibitory effect of FN on vWF:RCo binding to collagenmay explain our previousobservationthat the degree of adsorptionof vWF:RCo in normal plasma by collagen was lower than that of purified FVIII/vWF preparation(5). Effect of antibody treatment of FWP on the adhesion: To investigate the ibizty of the presence of?WorvWF:Konthe surface of FWP, FWP(4 x vs 10 /ml) were added by l/IO volume of rabbit anti-FN or anti-vWF:Ag and incubatedfor 48hr at room temperature.After FWP were washed three times with cacodylatebuffer, the adhesionto the collagencolumn was measuredand compared to control FWP which were added by normal rabbit serum. The adhesion of FWP to collagen(1,300ug) at 5 ml/min was 47.0 + 2.7%(anti-FN
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Fig. 4 Multimeric analysis of vWF:Ag of commercial MII. SDS agarose gel electrophoresis was performed for 24h at 10 mA at 16OC. The anode is at the bottom and the arrow indicates the top of the running gel. Samples; Lane 1, normal human plasma; lane 2, Hykryo; lane 3, Hemofil-HI'; lane 4, Kryobulin; lane 5, Confact-F; lane 6, Conco eight-HT; lane 7, Haemate-P. Fig. 5
_
COLLAGEN
CONCENTRATION(~g/ml)
Effect of FN on the binding of vWF:RCo to collagen. FN(125 ug/ml) was added to purified vWF(vWF:RCo 1 u/ml) and mixed with collagen(25-1,000 ug/ml). After the incubation of 1,000 rpm, 10 min at 37OC, the supernatant materials were assayed for residualvWF:RCo.The degree of binding of vWF:RCo to collagen in the presence of FN(closedcircle) was lower than that in the absence of FN(open circle).
treated FWP), 48.8 +0.8%(anti-vWF:Ag treated FWP) and 46.4 +1.3%(control Fwp). DISCUSSION In a previousstudy, we have describeda model for plateletadhesionto collagenand shown that the initialrate of adhesionof FWP to collagenwas increasedby vWF:Ag(6).A major purpose of the present study was to evaluate an effect of vWF and FN on platelet adhesion to collagen under different flow rate. In addition, we have studied an effect of FN on the binding of vWF:RCb to collagen. We have modified a previous method and used collagen immobilized beads, which allowed us to study platelet adhesion under differentflow rates. aggregation Under a scanningelectron microsoqq, no platelet-platelet was observedand most plateletsattachedto the collagenfibers, which were morphologicallysimilar to the contact plateletsin Baumgartnersystem (18), remainedunspreadedbecauseof the fixing of platelets with formaldehyde. Therefore,the adhesionof FWP in our system may be referredto the initial attachmentof the plateletsto collagen. VWF is necessaryfor initiationof normal plateletadhesionto the site
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of exposedvascularsubendotheliumand the role of vWF in plateletadhesion is well establishedespeciallyin the conditionsof high shear rates (3,19). However, there is a certain controversyover the effect; vWF increasesonly platelet contact(3,20),only platelet spreading(19)or both reactions(4). In our study describedhere, the purifiedvWF increasedthe adhesionof FWP to the collagen column only at the condition of high flow rates. By reducingthe flow rate to 0.5 or 1 ml/min, the promoting effect of vWF on adhesion can be markedly decreased. The adhesion was also high after pretreatment of the collagen column with vWF. These observations have corroborated the previous findings that decreased adhesion(initia1 attachment)of plateletsfrom patients with VWD is more pronouncedin high shear rate conditionsin Baumgartnerperfusionchamber experiments(18),and the impairedadhesionwas correctedby bound vWF to subendothelium(21). Sixma has described that partially reduced F'VIII/vWFincreases the adhesionof plateletsto human subendotheliumand postulatedthat multimeric size is not the only factor for supporting platelet adhesion(22).In our system, small or intermediate multimer of vWF:Ag did increase the FWP adhesion to collagentoo. This might be explainedby conformationalchange of vWF:Ag after binding to collagen, such as polymerizationof vWF:Ag. The multimer analysis of the samples of vWF:Ag bound to collagen is under investigation. FN is thought to be another glycoproteinwhich is involvedin plateletcollageninteractionand several investigatorshave reportedthat FN acts as platelet spreading factor(7,8), although oppositeobservationshave been reported by others(9,10). In our study, not only the adhesion of FWP but also the binding of vWF:RCb to collagen was stronglyinhibitedby FN. Our observations that the adhesion was inhibited by human plasma and the adhesion in the presence of commercial FVIII which contain large amount of FN was lower than that in the presence of purified vWF may also be explainedby the effect of EN. In respect to the inhibitoryeffect of IN on platelet-collagenor vWFcollagen,severalpossibilities should be considered. First, the binding site of collagen for FN is the same or very close to that for platelets. Secondly, small amount of the collagen binding proteins such as FN or vWF:Ag are adsorbedand remainingon the surfaceof FWP, which mediate the adhesion of FWP to collagens. This is, however, unlikely from the evidencethat pretreatmentof FWP with rabbit polyclonalantibcdies(anti-FN or anti-vWF:Ag)did not reduce the degree of adhesion and that FWP did not adhere to the denaturedcollagen(6). Thirdly, the binding sites of collagen for vWF:Ag and FN may also be very similar in each other as sharing for platelet receptor, glycoprotein IIb/IIIa after thrombin activation(23). Lastly, the inhibitionof the bindingof vWF:RCb and the adhesionof FWP to collagenby FN may be simply due to steric hindranceeffect. m understand the significance of the in vitro observation that different effects are present between FN and vWF on platelet-collagen interaction, further investigationsare required. In summary, the results introduced in this paper imply that the plateletadhesionto collagen may be partly controlled by vWF and FN. VWF promotes the adhesion only at high flow rate condition,and in contrast to platelet aggregationinduced by ristocetin,larger multimer of vWF:Ag may not be needed for the effect on adhesion. FN, on the other hand, acts as an inhibitorof platelet adhesion.
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We thank Drs HA Cooper and RH Wagner (Departmentof Pathology,Universityof North Carolina, Chapel Hill) for their invaluable suggestions. This work was supportedin part by a scientificresearchgrant(No.60770935)from the Ministry of Education,Japan REFERENCES CUNNINGHAM, L.W. The interactionof platelets with 1. SANTORO, %A. and collagen. In: Platelets in Biology and Pathology, vol -- 2. JL Gordonted.) Press, 1981, pp 249-264. New York, ElsevierNorthzolland Bioaical
2. PACKHAM, M.A. and MUSTARD, J.F. Platelet adhesion. 1n:Progressin Hemostasis and Thrombosis, vol -. 7 TH Spaet(ed.) Orlando, Grune and Stratton, 1984,~~ 211-288.
3. TURITTO, V.T., WEISS, H.J. and BAUMGARTNER, H.R. Decreased platelet adhesion on vessel segments in von Willebrandrs disease: a defect in initial plateletattachment. J. Lab. -Clin, Med. 102, 551-564, 1983. 4. LEYTIN, V.L., GORBUNOVA, N.A., MISSELWITZ, F., NOVIKOV, I.D., PODREZ, E.A.,PLYUSCH, O.P., LIKHACHOVA, E.A., REPIN, V.S. and SMIRNOV, V.N. Step-by-step analysis of adhesion of human platelets to a collagencoated surface defect in initial attachment and spreadingof platelets in von Willebrandrsdisease. ThrombosisRes. 34, 51-63, 1984. 5. MORIN, R.J., CHEN, A.F.T., NARAYANAN, A.S., RAYE, C., MOSS, R.A., SRIKANTAIAH,M.V. and BARAJAS, L. Platelet adhesion to collagen in normal and von Willebrandrsdisease subjects.ThrombosisRes. 17, 719-728,1980. Platelet-collagen 6. AIHARA, M., CoopER, HA. and WAGNER, RH. interaction: Increase in rate of adhesion of fixed washed platelets by factor VIII-relatedantigen Blood 63, 495-501,1984. 7. HOUDIJIK, W.P.M.and SIXMA, J.J. Fibronectin in artery subendothelium is importantfor plateletadhesion.Blood 65, 598-604, 1985. 8. KOTELIANSKY, V.E., LEYTIN, V.L., SVIRIDOV, D.D., REPIN, V.S. and SMIRNOV, V.N. Human plasma fibronectinpromotes the adhesionand spreading of plateletson surface coated with fibrillarcollagen.-FEBS L&t. 123, 5962, 1981. 9. SOCHYNSKY, R.A., BOUGHTON, B.J., BURNS, J., SYKES, B.C. and MCGEE, J.O.D. The effect of human fibronectin on platelet-collagen adhesion. 'Ihratbosis Res. 18, 521-533,1980. 10. MOON, D.G.,KAPLAN, J.E.and MAZURKEWICZ,J.E. The inhibitoryeffect of plasma fibronectinon collagen-induced plateletaggregation.Blood 67, 450457, 1986. 11. SARJI, K.E., STRATTON, R.D., WAGNER, R.H. and BRINKHOUS, K.H. Nature of von Willebrand factor:A new assay and a specificinhibitor. Proc. Natl. d---I Acad Sci. USA 71 2937-2941, 1974. -12. LAUREL& CB.
Quantitativeestimationof proteinsby electrophoresisin
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agarose gel containingantibodies. Anal. Bicchem.15, 45-52, 1966. 13. BINNIE, C., WAGNER, R.H., COOPER, H.A. and HERMANS, J. Parallel characterization of bovine von Willebrand protein (factor VIIIassociated protein) by light scattering and SDS gel electrophoresis. ThrombosisRes. 40, 523-532, 1986. 14. AIHARA, M., SAWADA, Y., UENO, K., MORIMOTO, S., YOSHIDA, Y., DE SERRES, M., COOPER, H.A. and WAGNER, R.H. Visualization of von Willebrand factor multimers by immunoenzymaticstain using avidin-biotin peroxidasecomplex.Thromb.Haemostas.55, 263-267, 1986. 15. BRASS, L.F. and BENSUSAN, H.B. The role of collagen quaternary 1480structurein the plateletzollageninteraction. --J. Clin. Invest.3 1487, 1974. 16. ALLAIN, J.P.,COOPER, H.A.,WAGNER, R.H.and BRINKHOUS, K.M. Platelet fixed with paraformaldehyde: A new reagent for assay of von Willebrand factor and platelet aggregating factor. --J. Lab. Clin. Med. 85, 318-328, 1975. 17. FAVILLE,R.J. and DAVIS, R.B. Measurement of plateletadhesiveness with collagencoated beads. ---J. Lab. Clin. Med. 68, 873-874,1966. 18. BAUMGARTNER,H.R The role of blood flow in platelet adhesion, fibrin depositionand formationof mural thrombi. Microvas.Res. 2, 167-179, 1973. 19. BOLHUIS, P.A., SAKARIASSEN, K.S., SANDER, H.J., BOUMA, B.N. and SIXMA, J.J. Bindingof factor VIII-vonWillebrandfactor to human arterial subendothelium precedes increased platelet adhesion and enhances J.Lab.Clin Med 97 568-576, 1981. platelet spreading. --d;-r
20. BOOYSE, F.M., FEDER, S. and QUARFOOT, A.J. Culture-produced subendothelium. II. Effect of plasma, F.VIIIR:WF and fibronectin on interaction of normal platelets with normal and von Willebrand porcineaortic subendothelium.Thrombosis Res. 28, 299-311, 1982. 21. SAKARIASSEN, K.S.,BOLHUIS, P.A. and Human blood SIXMA, J.J. platelet adhesion to artery subendotheliumis mediated by factor VIII-von Willebrandfactor bound to the subendothelium.Nature 279, 636-638,1979. 22. SIXMA, J.J.,SAKARIASSEN,KS., BEESER-VISSER,N.H.,CYITENHOF-ROVERS, M. and BOLHUIS, P.A. Adhesion of platelets to human artery subendothelium: Effect of factor VIII-von Willebrand factor of various multimeric composition.Blood 63, 128-139,1984. 23. HAVERSTICK, D.M., COWAN, J.F., YAMADA, K.M. and SANTORO, S.A. Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrandfactor substrates by a synthetic tetrapeptidederived from the cell-bindingdomain of fibronectin. Blood 66, 946-952, 1985.
EFFECT OF FIBRONECTINANDVON WILLEBRANDFACTORON THE ADHESION OF HUMAN FIXED WASHED PLATELEESTo COLLAGENIMMOBILIZEDBEADS
M. Aihara, H. Takami, Y. Sawada,S. Morimoto,K. Kariya, I. Kudo, K. Ueno, A. Kimura and Y. Yoshida First Departmentof InternalMedicineand Blood Transfusion Service,HirosakiUniversitySchool of Medicine, Hirosaki,Japan (Received 24.2.1986; Accepted in revi'sedform 11.8.1986 by Editor M. Matsuda)
The adhesion of human fixed washed platelets(FWP)to collagen was measured using collagen immobilized beads. The addition of normal plasma or severe von Willebrand disease (VWD) plasma to FWPd ecreased the adhesion, suggesting the presence of some inhibitors of platelet adhesion in human plasma. Although the adhesion of FWP in severe VWD plasma was not different from that of FWP in normal plasma, the additionof purified von Willebrand factor(vWF,l-2 u/ml ristocetin cofactor) to FWP in buffer increasedthe FWP adhesion at higher flow rates, and the percent of adhesion in the absence of vWF was 10 %(collagen 500 ug) and 30% kollagen 1,000 ug) of that in the presenceof vWF at 10 ml/min. The enhancing effect of the vWF on FWP adhesion was also observed by pretreatment of the collagen column with vWF suggesting the important role of bound vWF to the collagen; adhesion 72% to the collagen column(l,600ug) treated with vWF and 16 % to the collagencolumn without the pretreatment at 10 ml/min. The promoting effect of vWF was also present in some commercial factorVII1 preparations which had no large or intermediatemultimers of vWF antigen.The adhesionof FWP was inhibited by fibronectin(FN) and the binding of ristocetin cofactor(vWF:RoO)to collagen fiber was also inhibited by FN; bound vWF:RCoto 50 ug/ml collagenin the absence or presence of 125 ug/ml FN were 60% and 8% respectively. It is suggestedthat vWF, even small multimer of vWF:Ag, is involved in the initial platelet-collagen interactionat high flow rates, while plasma FN acts as one of anti-adhesionfactor. ------___-_-----_
Key words:Human platelets,platelet adhesion,collagen,fibronectinand von Willebrandfactor. Reprintsrequeststo Dr. M Aihara, 5 Zaifucho,Hirosaki036, Japan 661
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1NTR0DuCr1oN Adhesion of blood platelets to the site of vascular injury is one of the first steps in hemostatic plug formation. The reaction is generally considered to be the adhesion of platelets to exposed subendothelial connective tissues(l,2). There are several factors involved in this interaction, such as von Willebrand factor, fibrinogen, factor XIII, fibronectin and thrombospondin(2). The role of the von Willebrand factor(vWF) in platelet adhesion to subendothelium or collagen has been reported using several different technigues(3-5). We have previously reported that von Willebrand factor related antigen (vWF:Ag)increasesthe rate of adhesion of human fixed washed platelets(FWP) to collagen in suspension,and that high molecular weight forms of vWF:Ag preferentially bind to the fibrillarcollagen(6).However, it is still uncertainthat how vWF enhancesplateletadhesion to collagenand what kind of form of vWF is needed for the reaction. which is also high molecular weight Concerningabout fibronectin(FN), glycoproteinand involvedin platelet-subendothelium interaction,there are several different observations about an effect on platelet-collagen interaction. Although the requirement of FN for platelet adhesion to collagen has been described(7,8),inhibitory effect of FN on plateletcollagenhas been also reported by several groups(9,lO). In this paper, we describe a role of vWFand FN on platelet-collagen interaction.The adhesionof FWP to collagenwas inhibitedby normal plasma or plasma of the patient with severe von Willebrand disease(VWD). PurifiedvWF increasedthe adhesionof FWP to the collagenat a higher flow rate and an increased effect of vWF on adhesion was also observed after pretreatmentof the collagenbead column with vWF, suggestingthe important role of bound vWF in mediatingplateletadhesion.Furthermore,the adhesion of FWP to collagen was also increased by several commercial factor VIII preparations,which showed the lack of large or intermediate multimers of vWF:Ag. PurifiedFN, however, did inhibitnot only the adhesion of FWP to collagenbeads but also the bindingof vWF:RcO to collagenin suspension. MATERIALSANDMEX'HOIX
Materials: Reagents were allreagentgrade unless specified. Water was deionizedand glass distilled.Bovine serum albumin(BSA, crystallized and lyophilized) was obtained from Sigma Chemical Co.(StLouis), and human serum albumin(HSA)and human gamma globulin fraction were purchased from Green Cross Co.(Japan). Sepharose CL-4B was from Pharmacia Fine chemicals(Uppsala, Sweden). Agarose and Gel Bond Film for electrophoresis were from FMC Corp., Rockland, ME. sodium dodecyl sulfate(SDS, electrophoresis purity reagents)was from Bio-RadLabs, Richmond,CA. Human FN was purchased from Biomedical Tech., Inc., MA. and Cooper Biomedical Inc.,PA. The buffer used in the adhesionexperimentswas 0.05M caccdylateO.lM NaCl, pH 7.3(cacodylate buffer). Assay of vWF:RCo, vWF:Ag and FN:
The activity of vWF was measured as ristocetin cofactor(vWF:RCo) ssing a Payton dual channel aggregometer(Buffal0, NY). FWP(8 x 10 /ml), 0.2 ml was mixed with 0.2 ml of normal plasma dilutions or test materials in a siliconized cuvette and the mixture was then added by 0.2 ml of 4.5 mg/ml of ristccetin(H.Lundbeck Co., Copenhagen, Denmark). !Ihe initial slope of the aggregation curve was
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measured and used for the guantitation of vWF:RCo(ll).BothvWF:Ag and FN were measured by Laurel1 electroimmunoassay(l2) using 1% Seakem ME agarose and I:200 rabbit anti-human vWF:Ag which was prepared in our laboratoryand rabbit anti-humanFN(Behringwerke AG, Marburg,West Germany). ckleunit of these activitiesis, by convention,that amount found in 1 ml of pooled normal human plasma. Plateletpoor plasma: Blood was collectedwith informedconsentfrom twelve normal adult donors and one patient with severe VWD(vWF:RCo < 0.03 u/ml, vWF:Ag < 0.03u/ml) into1/9volumeof 3.2% sodium citrate. Platelet poor plasmas were centrifuged for 20 min at 3,000 rpm at 4OC, and then recentrifugedat 10,000rpm for 30 min at 4OC before stored at -7OOC CommercialMI1 preparations:CommercialfactorVIII preparationsused were Confact-F(Chemo-Sero-Therapeutic Res., Japan), Haemate-P(Hoechst, Div.; BoehringwerkAG), Hykryo(NipponSeiyaku Co.,.Japan),Hemofil-HT(Hyland Travenol Labs., USA), Conco eight-HT(Green Cross Co., Japan) and Kryobulin(Immuno AG.,.Austria). The ratios of vWF:RCo/vWF:Ag and the concentration of FN(u/ml) were 0.63, 1.9 in Confact-F, 0.59, 7.4 in Haemate-P, 0.49, 23.2 in Hykryo, 0.30, 30.2 in Hemofil-HT, 0.25, 29.1 in Conco eight-HT and 0.25, 8.8 in Kryobulin. For the adhesion experiments, each preparationwas diluted with cacodylatebuffer and the amount of vWF:Ag was adjustedto l-2 u/ml. Purified factor VIII/vWF complex: Human FVIII/vWF complex was purified from commercial FVIII preparation(Hykryo, Kryobulin) as previously described(l3). The commercial preparation was added by 25% polyethylene glycol to a final concentration of 5.5% and mixed for 30 min at 4OC. The precipitatewas collectedby centrifugationfor 30 min at 3,000rpm, at 4Oc and dissolved in 0.05M Tris-0.3MNaCl, 5 mM citrate, 50mM c-amino-caproic acid(EACA),pH 7.3. The material was adjustedat a concentrationof 5 mg/ml and was again precipitatedby polyethyleneglycol. The dissolved material was loaded onto Sepharose CL-4B equilibulated in the buffer and peak fractions in the voidvolume were pooled and dialyzed against cacodylate buffer. The supernatantmaterial after centrifugation 10,000 rpm for 30 min at 4OC was stored at -7OOC. vWF:Aq multimer: The multimeric forms of vWF:Ag were analysedby the method of SDS agarose gel electrophoresis using 1.5% Seakem HGT(P) agarose. The multimers were visualizedby immunoenzymatictechniqueusing avidin-biotin peroxidasecomplex as previouslydescribed(l4).Briefly,the gel was washed with distilledwater after the electrophoresis and reactedwith I:100antivWF:Ag in 1% human gamma globulin solutionovernight. The gel was washed with O.OlM phosphatebuffered saline(PBS)containing0.05% Tween-20, pH 7.4 and then incubatedwith l:lOO-200biotinylatedgoat anti-rabbitIgG. The gel was then incubated for 4hrs with avidin biotin-peroxidasecomplex(Vector labs. Inc.,Burlingame, CA).The peroxidase activity was visulalized with the substrate, 60 mg 4-chloro-I-naphthol(Bio-F&d), 0.06 ml of 30% hydrogen peroxidein 20 ml methanol-100ml PBS. Collagen& collagen coated glass beads: Dispersedbovine Achilles tendon collagen was obtained from Ethicon Inc., Somerville,NJ(a gift from Dr. RL Kronenthal).The material was shown to.bepolymeric.byelectronmicroscopic studies (15). The stock solution of collagen was prepared by adding the original collagen suspension (14 mg/ml) to the appropriate volume of distilled water. The mixture was rocked overnight at 4OC and then stored
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until used. 'Ibprepare collagencoated glass beads, 5ml of the collagen in distilledwater(250-2,000 ug/ml) was neutralizedby the addition of 0.8-1.0 ml of O.lM ammonium bicarbonate in a plastic box. Fifteen grams of glass beads(O.3-0.4 mm diameter, Eiken, Japan) were then added to the mixture and the mixture was vacuum dried overnightat room temperature. Fixed washedplatelets(FWP): FWP were prepared by a modification of the method-lain JP eta1 (16). Human vlatelets concentrates were washed with 6mM titrated saline; pH 7.1 and-were fixed with 1.8% formaldehyde solution, pH 6.4 overnight at 4'C. After fixing, the FWP were washed and then stored at 4OC, 8 x IO8 platelets/ml in 0.05M imidazole buffer containing ImM ethylene diamine tetraacetic acid(EJITA) and 0.02% sodium azide, pH 6.4. For the adhesion experiments, the FWP were resuspended in cacodylatebuffer. (luantitation of the adhesion--of FWP to collagenimmobilizedbeads: Adhesion -of FWP to collagen was tested by a column filtration method which was modified from an early system described by Fauville and Davis(l7). Approximately 3 gm of collagen coated glass beads were packed intoa 4 mm diameter vinyl chloride tube(Kohsin, Japan) under negativepressure. The FWP(1.5-2.0x 108/m1)ina IO mlsyringe were thenapplied to the collagen column at the constant flow rate using an infusionpump(MicroFeeder JP-S, Furue Science, Japan). Each 0.5 ml fractionwas collectedinto the plastic tubing and platelet count was measured by an electroparticle counter(Thrombocounter-C, Coulter, FL). The degree of adhesion was calculatedfrom the differencebetween the count of non adherentFWP in the passage through the column and that of initial FWP solution,and expressed as percent of the initial control. Because the adhesion was decreased by washing the collagencolumn with buffer prior to the applicationof FWP, the columns of dried collagenbeads were used in most experiments. Totestan ef ect of plasma, purifiedvWF, or FN on FWP adhesion to collagen,FWP(4 x IO6/ml) in buffer containingl-2% BSA was mixed with equal volume of test materialsand used for the adhesionexperiment. Morphologicalobservationof the adherent platelets was performed by scanningelectron microscopy. The collagen bds were removed from the column after the adhesionexperimentand prefixedwith 1% glutaraldehydein O.?M phosphate buffer, pH 7.4 overnight. After washing the beads with phosphatebuffer, the samples were post-fixedwith 1% osmic acid(WakoPure Chemical Ind.,Japan) for 2-3 hours and then dehydrated by acetone. The materials finally evaporated with carbon and gold were observed by a scanningelectronmicroscope(Nippon Eenshi, Japan). Binding experimentswith collagen: Normal human plasma, 0.3 ml was mixed with 0.3 ml of the collagen suspension in a siliconized aggregometer cuvette. The mixture was stirred at a setting of 1,000 rpm for IO min at 37OC using a Payton aggregomter. The supernatant, after removal of the collagenaggregatesby centrifugationfor 5 min at 3,000 rpm, were assayed for residualactivitiesof vWF and FN. Binding of vWF to the collagen was also evaluated using purified vWF(4 u/ml vWF:RCo) and the effect of FNon vWF adsorptionto collagenwas tested.After two millilitersof the purified vWF was mixed with 2 ml of 500 ug/ml FN or cacodylatebuffer, 0.3 ml of the mixture was then added by equal volume of collagen in suspension. The supernatant materials after the centrifugation were assayedin a similar manner.
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RESULTS FWP adhered to collagen coated glass beads without platelet aggregation(Fig 1). Most platelets attached to the collagen fibers were unspreadedand their patternswere similar to those of the contactplatelets accordingto the definitionby Baumgartner(18). Adherence--of FWP to collagen coated glass beads: In the control experiment using vinyl chloride tubing and glass beads without collagen, FWP in caccdylatebuffer did not adhere to the tubing, but did adhere to the glass beads. The number of platelets passed through the control glass beads column(30cm) was 2.6% in first fraction,6.8% in second fraction,17.0%in third fraction and 30.0% in forth fraction at the f ow rate of 1 ml/min, when compared to the originalplateletcount(2.0x 10B/ml). This nonspecificadhesionwas partly decreased by either increasing the flow speed or increasingthe size of glass beads in the column(datanot shown here). Bovine serum albumin(BSA) also inhibited the nonspecific binding of FWP to the glass beads without collagen. When FWP resuspendedin BSA at the concentration between 0.5% and 5%, the binding of FWP to the glass beads was completelyabolishedat the concentrationof BSA above 0.5%. Therefore, cacodylate buffer with 0.5% BSA was used in the adhesion experimentswith the collagencoated glass bead column. Effect of ----flow rate on the FWP adhesionto collagen: FWP were applied to theiiagen column(the amount of cXlagen, 1,000ug/column) and each 0.5 ml fractionwas collected. At the flow rate of 1 ml/min, the percent of adhesion in the fraction from first to sixth were 67.9%, 61.7%, 55.5%, 48.8%, 46.0%,and 42.0% respectively(Fig.2). At a higher flow rates such as 5 or 10 ml/min, however, the percent adhesion in each fraction was constant from the first 0.5 ml. To evaluate aneffectof the flow rate or collagenconcentrationon the FWP adhesion,percentplateletadhesionto the collagen column was arbitrarily defined as the mean values of four
FIG. 1 Adhesionof FWP to collagen.FWP were mixed with collagenin suspensionor collagen immobilized beads, and the mixtures were observed by scanning electron microscopy. (A) collagen in suspension(bar 1 urn), (B) collagen coated glass beads(bar5 um).
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FIG. 2 Effect of flow rate on the adhesion of FWP to collagen.Conditions: platelet 1.5-2.0 x log/m1 in caccdylatebuffer with 0.5% BSA; collagen, 1,000 ug/column;fractionsize, 0.5 ml; flow rate, l(A), 5(B), 10(C) ml/min. The degree of adhesion (mean value of first 4 fractions) were 58.5% (A), 24.4%(B) and 13.1%(C) respectively.
FIG. 3
250
500
COLLAGEN
750
1000
CONCENTRATION
”
2000
IpglColumnl
Effectof collagenconcentration on the platelet adhesion. Conditions were the same as in Fig. 2 except that the amount of the collagen in column was varied from 250 to 2,000 ug. The percentadhesion increased with an increasing amount of collagen in the column at all flow rates, but decreased with increasing flow rate at the constant amount of collagen.
fractions(lst-4th,2ml). The effects of varied amounts of collagen were tested at 1, 5 and 10 ml/min flow rates. The percent of plateletadhesion increasedwith an increasingamount of collagen in the column at all flow rates(Fig. 3). With a given amount of collagen in a column, the percent adhesion decreased with increasing flow rate. Percent adhesion to the column containing 500 ug collagen was 40.120.8% at Iml/min, 9.5+ 1.2% at 5ml/min and 3.8+ 0.8%at IO ml/min(mean+ SD, n=3). Effect of vWF on FWP adhesion to collagen : -----
When purified vWF was introducedinto the test system, thYadhesion of FWP to collagencolumn was significantly increased. With the glass bead column containing 1,000 ug collagen,the percent adhesion in the absence of vWF was 58.5%at the flow rate of 1 ml/min, while 76.5% adhesion was observed in the presence of vWF(lable1). The enhancingeffect of vWF on adhesion increasedwith flow rate; adhesion in the absence of vWF was 10.9%(collagen 500 ug) and 30.2%(collagen1,000ug) ofthevalue in the presence of vWF at IO ml/min. The effect of vWF on adhesion was dose dependent and the percent adhesion(500 ug collagen) was 78.5%(vWF:RCo 4u/ml), 64.8%(2 u/ml) and 24.8%(1u/ml) at 5 ml/min. The effect of vWF on FWP adhesion to collagencolumn was also tested by pretreatment of the column with vWF. The collagen coated glass bead column(30cm length, 1,600 ug collagen)was previouslywashed with 5 ml of either buffer or purifiedvWF(vWF:ROo4-5 u/ml) at a constantflow rate of 2
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TABLE I Effect of Purified VWF on FWP Adhesion to Collagenat different Flow Rate(Collagen 1,000 ug/column;final concentration of vWF, I-1.5u/mlvWF':RCo; Value, I&an + SD, n=3, *p
Without
VWF
With
VWF
1
58.5 + 1.3* 76.5 5 1.4 5 24.4 T 1.6* 60.0 2 1.0 10 13.1 z 2.6" 43.2 2 2.0 -__--_--_---_--__--_--~---_--~--~_--~--~~--_--~------~------~---------~-
TABLE II Effectsof mmercial MI1 Preparationson FWP Adhesionto collagen 3 /ml were mixed with test materials,controlbuffer; (FWP 4 x 10 cacodylatebuffer containing1% EEA, Value; mean 2 SD, n=3, ND: not done, P
collagen
500 uglcolumn 2000 ug/column (5 ml/min flow rate) (IO ml/min flow rate) -~--------------------_--~---_--_------_------_---_--_--__--__-_________ controlbuffer 6.0 + 0.5 29.4 + 3.2* confact-F 14.9 T 2.9** 55.5 71.7" Haemate-P 17.0 + 0.6* 61.8 + 0.8" Hykryo 6.7 7 2.3 47.6 7 0.4* Conco eight-HT 10.0 T 0.1* 54.7 T 0.4* Hemofil-HT 6.8 T 0.3 63.8 T 1.7* Kryobulin 5.6 7 0.8 44.6 + 0.3* PurifiedvWP 24.8 z 1.9* GD -_--_------_-_--_-__-~---~~-~----~-~~---~-~------~--~------~------------
ml/min. The solution remaining inside the column was then removed by passing sufficientair at a flow rate of 10 mllmin. The adhesion0 FWP to collagen was then examined by filtering the FWP(1.5-2.0 x 10B /ml in caccdylatebuffer without RSA) through the collagencolumn at flow rates of 0.5, 1, 3, 5 and 10 ml/min. Each 0.5 ml fraction was collected, but the first 5 drops were excluded to minimize the dilution of FWP in the first fraction because of the wet column. The percent adhesion of FWP to the collagen column pretreatedwith vWF was higher than that of the untreated collagen column, especially at high flow rates; the adhesion to the column without pretreatmentwas 93.8% at 0.5 ml/min, 75.7% at 1 ml/min, 54.7% at 3 ml/min, 51.6% at 5 ml/min and 16.0% at 10 ml/min, while the adhesion to the column with pretreatmentwas 92.4%at 0.5 ml/min, 95.0% at 1 mllmin, 94.1% at 3 ml/min, 87.9% at 5 ml/min and 71.9% at 10 ml/min(meanof duplicate).
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Effect of commercial FVIII preparations -on FWPadhesionto collagen: The Be& patterns of six commercialFVIII preparationsw= shown in Fig. 4. All preparationswere characterizedby lack of large multimer of vWF:Ag and furthermore,four except confact-Fand Haemate-P showed the absence of intermediate multimer. The triplet structure of each small multimer was abnormal in all preparations and the fastest migrating subband of the triplet was increased in quantity, while the central band was stained dominantlyin normal plasma. When FWP were added by these preparationsin which vWF:Ag was adjusted to 1 u/ml, the degree of adhesionto collagen(500 ug) at 5 ml/min was significantly increased by Confact-F, Haemate-P and Conco eight-HT,although the enhancingeffects on adhesion were lower than that of purified vWF(Table II). However, when the concentrations of the preparationswere increasedto twice(vWF:Ag2 u/ml), the adhesion measured using 2,000 ug collagen at IO ml/min was increased by all preparations. Effect of plasma -on FWP adhesion to collagen: When FWP(4 x 108/ml) were -mixed with equal volume of normalplasma dilutions, the FWP adhesion to collagendecreasedby increasingthe plasma concentration;the adhesion to collagen column(20 cm, 1,300 ug collagen) was 45.5% in the absence of plasma and 26.0 % (I:2normal plasma), 29.7%(1:4), 44.7%(1:8),46.5%(1:16) and 45.2% (1:32)at 5 ml/min. The inhibitory effect of plasma on FWP adhesionwas also observedby severevWD plasma and the adhesionof FWP in VWD plasma(18.3+1.6%)was not significantlydifferent from that of FWP in normal plasma(21.0-+ 0.8%),even at 5 ml/min flow rate. Effect of FN on FWP adhesionto collagen:When purifiedFN was added to FWP, theadhesionofFWP to collagen column!1300 us) was inhibited dose dependently; the adhesion was 34%(FN, 31 ug/ml), 29%(62 ug/ml), 16%(125 ug/ml), 2%(250 ug/ml) and 51%(intheabsence of ~~)at 5 ml/min flow rate. The inhibitory effect of FN on the adhesion was also observed at 0.5 and 1 ml/min flow rate, and the adhesion was decreasedafter pretreatmentof the collagenby purified F~(datanot shown). Binding of vWF to collagen in suspension: Both FN and vWF:Ag in normal human plasma-a to collagen in suspensionand the dwee of binding was increased by increasing collagen concentration(data not shown). When purified vWF(4 u/mlvWF:RCo) was mixed with 500 ug/ml FN and tested for collagen binding experiment, the residual amount of vWF:RCo in supernatant was higher in the presence of FNthanthat in the absence of FN;the amount of bound vWF:RCo to the collagen(fina1 cont. ug/ml) was 100%(200 and 500 ug/ml), 40%(100 ug/ml), 28%(50 ug/ml)and nil(25 ug/ml) inthe presence of FN; however, 100%(100, 200 and 500 ug/ml), 60%(50 ug/ml)and 32%(25 ug/ml) in the absence of FN. The degree of binding of vWF:RCo in the presence of FN was approximately13% of that in the absence of FN at the concentration of the collagen 50 ug/ml(Fig. 5). This inhibitory effect of FN on vWF:RCo binding to collagenmay explain our previousobservationthat the degree of adsorptionof vWF:RCo in normal plasma by collagen was lower than that of purified FVIII/vWF preparation(5). Effect of antibody treatment of FWP on the adhesion: To investigate the ibizty of the presence of?WorvWF:Konthe surface of FWP, FWP(4 x vs 10 /ml) were added by l/IO volume of rabbit anti-FN or anti-vWF:Ag and incubatedfor 48hr at room temperature.After FWP were washed three times with cacodylatebuffer, the adhesionto the collagencolumn was measuredand compared to control FWP which were added by normal rabbit serum. The adhesion of FWP to collagen(1,300ug) at 5 ml/min was 47.0 + 2.7%(anti-FN
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Fig. 4 Multimeric analysis of vWF:Ag of commercial MII. SDS agarose gel electrophoresis was performed for 24h at 10 mA at 16OC. The anode is at the bottom and the arrow indicates the top of the running gel. Samples; Lane 1, normal human plasma; lane 2, Hykryo; lane 3, Hemofil-HI'; lane 4, Kryobulin; lane 5, Confact-F; lane 6, Conco eight-HT; lane 7, Haemate-P. Fig. 5
_
COLLAGEN
CONCENTRATION(~g/ml)
Effect of FN on the binding of vWF:RCo to collagen. FN(125 ug/ml) was added to purified vWF(vWF:RCo 1 u/ml) and mixed with collagen(25-1,000 ug/ml). After the incubation of 1,000 rpm, 10 min at 37OC, the supernatant materials were assayed for residualvWF:RCo.The degree of binding of vWF:RCo to collagen in the presence of FN(closedcircle) was lower than that in the absence of FN(open circle).
treated FWP), 48.8 +0.8%(anti-vWF:Ag treated FWP) and 46.4 +1.3%(control Fwp). DISCUSSION In a previousstudy, we have describeda model for plateletadhesionto collagenand shown that the initialrate of adhesionof FWP to collagenwas increasedby vWF:Ag(6).A major purpose of the present study was to evaluate an effect of vWF and FN on platelet adhesion to collagen under different flow rate. In addition, we have studied an effect of FN on the binding of vWF:RCb to collagen. We have modified a previous method and used collagen immobilized beads, which allowed us to study platelet adhesion under differentflow rates. aggregation Under a scanningelectron microsoqq, no platelet-platelet was observedand most plateletsattachedto the collagenfibers, which were morphologicallysimilar to the contact plateletsin Baumgartnersystem (18), remainedunspreadedbecauseof the fixing of platelets with formaldehyde. Therefore,the adhesionof FWP in our system may be referredto the initial attachmentof the plateletsto collagen. VWF is necessaryfor initiationof normal plateletadhesionto the site
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of exposedvascularsubendotheliumand the role of vWF in plateletadhesion is well establishedespeciallyin the conditionsof high shear rates (3,19). However, there is a certain controversyover the effect; vWF increasesonly platelet contact(3,20),only platelet spreading(19)or both reactions(4). In our study describedhere, the purifiedvWF increasedthe adhesionof FWP to the collagen column only at the condition of high flow rates. By reducingthe flow rate to 0.5 or 1 ml/min, the promoting effect of vWF on adhesion can be markedly decreased. The adhesion was also high after pretreatment of the collagen column with vWF. These observations have corroborated the previous findings that decreased adhesion(initia1 attachment)of plateletsfrom patients with VWD is more pronouncedin high shear rate conditionsin Baumgartnerperfusionchamber experiments(18),and the impairedadhesionwas correctedby bound vWF to subendothelium(21). Sixma has described that partially reduced F'VIII/vWFincreases the adhesionof plateletsto human subendotheliumand postulatedthat multimeric size is not the only factor for supporting platelet adhesion(22).In our system, small or intermediate multimer of vWF:Ag did increase the FWP adhesion to collagentoo. This might be explainedby conformationalchange of vWF:Ag after binding to collagen, such as polymerizationof vWF:Ag. The multimer analysis of the samples of vWF:Ag bound to collagen is under investigation. FN is thought to be another glycoproteinwhich is involvedin plateletcollageninteractionand several investigatorshave reportedthat FN acts as platelet spreading factor(7,8), although oppositeobservationshave been reported by others(9,10). In our study, not only the adhesion of FWP but also the binding of vWF:RCb to collagen was stronglyinhibitedby FN. Our observations that the adhesion was inhibited by human plasma and the adhesion in the presence of commercial FVIII which contain large amount of FN was lower than that in the presence of purified vWF may also be explainedby the effect of EN. In respect to the inhibitoryeffect of IN on platelet-collagenor vWFcollagen,severalpossibilities should be considered. First, the binding site of collagen for FN is the same or very close to that for platelets. Secondly, small amount of the collagen binding proteins such as FN or vWF:Ag are adsorbedand remainingon the surfaceof FWP, which mediate the adhesion of FWP to collagens. This is, however, unlikely from the evidencethat pretreatmentof FWP with rabbit polyclonalantibcdies(anti-FN or anti-vWF:Ag)did not reduce the degree of adhesion and that FWP did not adhere to the denaturedcollagen(6). Thirdly, the binding sites of collagen for vWF:Ag and FN may also be very similar in each other as sharing for platelet receptor, glycoprotein IIb/IIIa after thrombin activation(23). Lastly, the inhibitionof the bindingof vWF:RCb and the adhesionof FWP to collagenby FN may be simply due to steric hindranceeffect. m understand the significance of the in vitro observation that different effects are present between FN and vWF on platelet-collagen interaction, further investigationsare required. In summary, the results introduced in this paper imply that the plateletadhesionto collagen may be partly controlled by vWF and FN. VWF promotes the adhesion only at high flow rate condition,and in contrast to platelet aggregationinduced by ristocetin,larger multimer of vWF:Ag may not be needed for the effect on adhesion. FN, on the other hand, acts as an inhibitorof platelet adhesion.
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We thank Drs HA Cooper and RH Wagner (Departmentof Pathology,Universityof North Carolina, Chapel Hill) for their invaluable suggestions. This work was supportedin part by a scientificresearchgrant(No.60770935)from the Ministry of Education,Japan REFERENCES CUNNINGHAM, L.W. The interactionof platelets with 1. SANTORO, %A. and collagen. In: Platelets in Biology and Pathology, vol -- 2. JL Gordonted.) Press, 1981, pp 249-264. New York, ElsevierNorthzolland Bioaical
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