Abstracts of papers presented at the Annual General Meeting of the British Society for Haemostasis and Thrombosis, University of Nottingham, 19-20 March 1992
I LZ! A STRUCTURAL-FUNCTIONAL STUDY OF PLATELET
fLlTHROHBTN CENERATJON: A PIVOTAL EVENT IN THE REGULATION OF HEMOSTASIS. Alan R. Giles. Departments of Pathology 6 Medicine. Queen's University. Kingston. Ontario. Canada.
MEMBRANE GLYCOPROTEIN CD36. JL McGrePor, HE1 Ghissasi. L McGregor. B Catimel. INSERM U331. Institute Pasteur. Faculte de medecine Alexis Carrel. F-69372 Lyon, France.
One o f the rmerging dilemnas in our understanding of thq regular ion ot hemastasis rs the ubiquitous nnture of thrombin's activity. For example. on one hand it appears to be essential for its own generation via activation of the essential cofactors V and VIII and yet its activity is limited by its ability to generate activated prorrin C (APC). Failure in its generation, p p . hemophilia A and B . results in a devastating hemostatic deficit whereas inappropriate generation, eg. in DIC. may be equally devastating and frequently much wrc difficult to manaRe. Although the medical literature is replete with detailed characterizations o f individual aspects of thrombin's activity. there 1s a major nerd to ent.ablish and characterize the mechanisms by which these interacting and divergent processes are regulated in Y I V O . W e now have considerable experience with an animal model that shows promise in asfiistinr: rhis process. A procoagulant stimulus is developed in normal or hemstatlcilly compromised animals by the intusion of two components o f the prothrombinase complex, factor Xa and procoagulant active phospholipid in the form of vesicles comprised o f phosphatidylcholine/phosphat.idvlserine (PCPS). This combination has been shown to induce a positive hemostatic efFect in bypassing factor VIII and correcting the bleeding diathesis in factor VIII deficient animals. However, the response depends on the absolute and relative concentration of each component to the other. Paradoxically. manipulation of this may induce a bleeding diat,hesis in normal animals. We have monitored the effects ~ n d u c e d on a number of hemostatic mechanisms including von Willrbrand factor. protein C and the fibrinolytic system. Substantla1 changes occur in all three systems that would promote both procoagulant and anticoagulant activities. The mechanisms that dlctate as to whether a net negative or porit.ive h m m s t a t i c rrsult ensues remain to be fully elucidated. However, the availability of coagulant active phospholipid emerges as an essential issue in this outcome.
Clycoprotein CD36. also known as GpIllb or CplV. is a major glycoprotein (GP) present on platelets and also on monocytes. melanomas and endothelial cells. CD36 plays an important role in platelets as one of the receptors to thrombospondin (TSP) and collagen. Moreover. recent studies have shown that Plasmodium falcioarum infected erythrocytes bind to purified CD36. The site on CD36 that interacts with TSP. collagen or malaria infected erythrocytes remains to be determined. In this study plots of hydrophilicity. hydrophobicity. alpha-helices. betasheets. segment mobility and acrophilicity were ?erformed on CD36 (Appligene. Strasbourg). Ten 'iydrophylic peptides spanning the entire amino acid sequence. deduced from the cDNA sequences of CD36, were selected in order of decreasing antigenicity and synthesized using Fmoc chemistry on an Applied Biosystems 431A. Three peptides Pa. Pb. Pc were found to bind to purified TSP using an ELISA system. The binding of TSP to peptide Pb. but not Pa, is inhibited by EDTA. Peptide Pa showed 55% less binding to TSP than peptide Pb. Peptide Pa when added at 200pM. prior to stimulation of platelets by thrombin (0.2-0.4Ulml). inhibited respectively aggregation and endogenous TSP binding. These studies indicate that the platelet CD36 molecule may have two different sites involved in interacting with thrombospondin.
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British Society for Haemostasis and Thrombosis
I L ~ C E L L U L A INTERACTIONS R WITH HEPAILIN:NEW PERSPECTIVES ON AN OLD DRUG. ,J P BiPnnon. A Minter, CN Chesterman. Heart Research Institute and Prince of Wales Hospital, Sydney, Australia. Although the anticoagulant activity of heparin is evidenced by its ability to enhance the activity of ATIII and prevent the clotting of plasma, it is becoming increasingly clear not only that a range of cellular elements are crucial to thrombosis but also that heparin is a multifunctional drug. I n this context we are examining its effects on the cells in the vascular tree, and its ability to modulate the synthesis and release of entdothelial products relevant t o coagulation and fibrinolysis. Human umbilical vein endothelial cells (HUVEC) were grown for 6 days with or without heparin and endothelial growth factor (ECGF). These supplements dramatically inhibited both the basal and thrombin challenged release of prostacyclin (PGI2), plasminogen activator inhibitor 1 (PA1- 1) and thrombospondin (TSP), and and stimulated synthesis of of tissuetype plamnincgen activator (t-PIi) and von Willebrand factor (vWF) . Both supp1ement;s were required for some of these responses, consistent with the effect of heparin being mediated through stabilisation or stimulation of the function of ECGF. However, heparin was able to modulate TSP and vWF release in the absence of exogenous ECGS, which implies a direct mechanism of control by heparin. In general these effects of heparin could stimulate platelet activation and adhesion, and promote fibrinolysis. They are also the opposite of 'those reported to be generated by inflammatory mediators such as interleukin 1 (IL-1). Preconditioning of H W E C with heparin and ECGF counteracted the effects of IL-1 on PGI2 and vWF release. In addition we have used a model system in which H W E C are cultured on filters to demonstrate that both unfractionated and LMW heparin reduce the permeabilit!{ of the endothelial monolayer t o plasma proteins. Such activities may contribute to a physiologically relevant antii n f l m t o w activitv.
OT IDENTIFICATION OF ANTITHROMBIN d i N A IN PERIPHERAL BLOOD LYMPHOCYTES AND ITS USE IN THE CHARACTERISATION OF ANTITHROMBIN VARIANTS. P e w DJ. Carrel1 RW. Dcpt. of Haematology. University of Cambndge. MRC Ccnue. Hills Rd.. Cambridge. CB2 2Q.H.
The charactensation of the dysfunctional variants of many proteins has proved: invaluable in identifying key residues involved in heir function. Despite advances in rapid screening techniques, he complexity of many genes makes the idennfication of DNA mutations a daunang task. A more logical approach would be to analyse the mRNA for a specific pirotein and although for many proteins mRNA is limited to specific organs cg Annthrombin (AT) and the liver, there is now increasing evidence that small amounts of a correctly spliced trmscnpt for vanous tissue-specific gerles can be detected i n a wide vanety of non-\pecific cells. We have developd a melhod for detecting AT mRNA in penpherd blood lymphocytes and show its value in the idennfication 01 AT v;inmts. Total cellular RNA was isolated from penphen.1 blood lymphocytes. A senes of nested oligonucleotide pnmers were designed to amplifv the entre AT cDNA in two overlapping fragments of X03bp :md 760bp: The extreme downstream oligonucleotides were used as pnniers for the reverse uanscnption. Following reverse aanscnption two separate PCR amplifications were pertomed using the nested pnmer!;. After the first PCR, no bands were seen but atter the second PCR. fragments of the expected sizes were clearly visible. Rcstnction mapping of these 2 fragments confumed them to be pans of the AT cDNA. More recently we have shown that it is possible to obtain a full length AT cDNA by modifying the PCR parameters and using the extreme 5' md 3' pnmers in the amplification reaciions.
In order to evaluate this approach i n the identification of AT vmants. total RNA was isolated from a dysfunctional vanant (AT Brighton. 281 lie-Am). reverse uanscnbed and amplified. The product was punfied and directly sequenced. A single additional band was observed corresponding to a T-A subsnturion and confirming the mutation previously identified at the genomic level tDaly et al: unpublished). We have demonstrated that AT mRNA is present in penpheral blood lymphocytes. that i t can be readily amplified. This approach provides a simple and rapid method for the detection of mutations within the AT gene and should prove useful i n the charactensarion o f diverse genetic diborders.
02A RECURRENT 6 BASE PAIR DELETION IN THE ANTITHROMBIN GENE IDENTIFIED BY DNA HETERODUPLEX DETECTION B.J. Ol&. D. A. Lane. C. H. Beresford. U. Abildgaard. S . L. Thein. Institute of Molecular Medicine, John Radcliffe Hospital. Oxford; Depanment of Haematology, Channg Cross and Westminster Hospital Medical School, London; Depanmenr of Pathology, University of Otago Medical School. Dunedin, New Zealand; Depanment of Medicine, Aker Hospital. Oslo. Norway.
In 'ype la antithrombin deficiency. i n which plasma levels of the inhibitor measured by immunological assays approximate 50% of normal, the abnonnality in the majority of individuals appears to be a null allele resulting from a point mutation. As p a n of the stategy for the investigation of the genomic basis of these deficiencies we screened for the presence of DNA hcteroduplexes i n amplified brgments of the gene spanning the antithrombin protein coding regions. Hetercduplexes with a similar pattern of altered mobility were identified i n two families with type la antithrombin deficiency, in the region of exon 3A. The amplified products were directly sequenced in each of the kindreds and sequence analysis showed identical 6 base pair deletions within this region; haplotype analysis of the mutant alleles confinned that the deletions had probably arised independently i n the two families. The deletion occurred within the sequence TIT AAG ? T T (codons 106-lox). removing either TTT AAG or AAG TTT.The deletion site is clearly tlanked by a repeat sequence (TIT.and the complete removal of one repeat and the intervening bases (AAG) is compatible with slippage and mispairing at the repeat site dunng DNA replication as a mechanism for the deletion. Although the mutation removes two intact codons and does not result in a frame shift in trmslation a mutant protein could not be detected in the plasma. It may be that the predicted variant protein is highly unstable. as the deleted bases encode residues which form the turn at the end of helix C in the tertiary structure models of the serpins. and the hydrophobic F108 is predicted to lie in the inienor of the protein.
03 INHERITED ATIII
DYSFUNCTION CAUSED BY THE INTENSIVE EXPRESSION OF AN OTHERWISE NORMAL PLASMA COMPONENT VS Lindo, E M e l i s s a r i , M Learmonth, DN Cooper, W Kakkar. Thrombosis Research I n s t i t u t e , Emmanuel Kaye Building, Manresa Road, London SW3 6LR.
A f a m i l i a l , f u n c t i o n a l ATIII d e f e c t was s t u d i e d i n t h r e e members of a l a r g e family who s t a r t e d s u f f e r i n g severe venous thromboembolism a f t e r the age of 18. Both anti-Xa and antithrombin heparin cofactor a c t i v i t i e s were decreased a t 60% of normal, whereas progressive ATIII a c t i v i t y was found t o be reduced a t 4 5 % . I n c o n t r a s t , ATIII a n t i g e n l e v e l s were normal. Crossed immunoelectrophoresis of p a t i e n t s plasma a g a i n s t antiserum t o human ATIII, i n t h e presence and absence of heparin, was no d i f f e r e n t t o t h a t of t h e c o n t r o l . ATIII p u r i f i c a t i o n by dextran s u l p h a t e p r e c i p i t a t i o n followed by heparin seeharose a f f i n i t y chromatography and e l u t i o n by NaCl g r a d i e n t (0.15-2.OM), showed a normal p r o t e i n recovery and e l u t i o n P r o f i l e , i n a d d i t i o n t h e p u r i f l e d ATIII f r a c t i o n s showed normal progressive a c t i v i t y , heparin c o f a c t o r and anti-Xa a c t i v i t i e s . The p a t i e n t s ' p u r i f i e d ATIII a l s o had a normal mobility on SDS-polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) and formea an SDS s t a b l e complex with p u r i f i e d human thrombin. In a d d i t i o n , CNBr cleavage of t h e p u r i f i e d p r o t e i n followed by HPLC a n a l y s i s and SDS-PAGE d i d not show any abnormal fragments. PAGE, however, of p a t i e n t s plasma on 7 . 4 % n a t i v e g e l s , immunoblot and probing with antiserum t o human ATIII followed by enhanced chemiluminescence ( E C L ) d e t e c t i o n , showed t h e presence o f an i n t e n s e l y immunoreactive band which was not seen i n t h e p u r i f i e d ATIII o r i n p a t i e n t s ' plasma i n t h e presence of SDS. Ferguson p l o t a n a l y s i s showed t h e band t o have a .W of approximately 1 9 4 , 0 0 0 Da. Inununob l o t and ECL a n a l y s i s of 1 2 o t h e r p a t i e n t s ' plasma ( A T 1 1 1 a n t i g e n s 110-160%) d i d not show t h i s i n t e n s e band. Further i n v e s t i g a t i o n s revealed t h a t t h e ATIII a c t i v i t y recovered only when t h e band was removed through t h e p u r i f i c a t i o n Process although no loss of ATIII antigen had occurred. We t h e r e f o r e conclude t h a t t h i s i n h e r i t e d ATIII dysfunction i s caused by t h e i n t e n s i v e expression of an otherwise "normal" plasma component a c t i n g a s an " i n h i b i t o r " , because i t s removal through t h e p u r i f i c a t i o n process r e s u l t s i n 100% recovery of f u n c t i o n a l a c t i v i t y of ATIII and because gene analvsls has Shown no abnormalities i n the ATIIL a ~ u e
Abstracts 04
FIAEhIOPHILIA A DIAGNOSIS B Y ANALYSIS OF A NOVEL DINUCLEOTIDE TANDEM REPEAT SEQUENCE WITHIN THE FACTOR VllIGENE. M.R.A. Lalloz, J . H . McVey, K . MichdeLides and E . G . D . Tiiddenham. Haemostasis Research Group, Clinical Researvh Centre, Harrow, Middx. H A 1 3UJ, U . K . The factor VlIl (FVIII) gene comprises 26 exons spanning 186Kb of DNA located a t t h e distal e n d of the long arm of the Xchromosome. Defects in this gene cause haemophilia A , a bleeding disorder affecting 1:5000 males. Restriction fragment length polymorphisms ( RFLPs) used in c a r r i e r detection a n d prenatal diagnosis a r e informative in only 75% of cases. We recently reported a highly informative ( > g o % ) multi-allelic dinucleotide tandem repeat. (CA),, specific to intron 13of t h e gene (Lallozet a1 1991. Lancet 3 3 8 , 207-11). We have identified and characteriseda new dinucleotide repeat of the form (CA),(TC),, locahsed b y wnventional mapping to intron 22 of the gene. Primers to IINA sequence flanking the intron 22(CA).,(TC).. repeat were i!hosen to amplify genormc DNA samples using conditions for polymerase chain reaction ( P C R ) similar to those used f(rr the intron 13(CA), r e p e a t . Primer specificity was demonstrated by t h e presence of bands corresponding to intron 13( C A ) , . and 2 2 ( CA),( TC).. repeats a f t e r simultaneous PCR of normal genonuc D N A but absence of the latter band only when patient DNA with a deletion mutation s p a n m n g exons 14 to 26 of t h e FVIII gene w a s amplified u n d e r identical conditions. Two intron 22(CA).,(TC)..allelic b a n d s were observed. one comprising 2 5 , the other 26 dinucleotide repeating units. Family studies showed Mendelian inheritance with allelic frequencies ( 5 5 Xchromosomes analysed) of 33% for t h e s h o r t e r and 67% for t h e longer repeat. The iritron 22(CA)..(TC)., repeat tracks with haemophlia A in family studies. The c a r r i e r s t a t u s of two s i s t e r s previously indistinguishable because of identical haplotypes is resolved (one normal the other a n informative c a r r i e r ) following analysis of t h e intron 2 2 repeat. The occurrence of a spontaneous mutation in a three grneration family s t u d y is confirmed to be grandpaternal in origm a f t e r analysis of this r e p e a t . Similar analysis of a chorionic villus sample revealed a n unaffected male fetus to be carried b y a female otherwise uninformative f o r all intragenic FVIII markers analysed . Simultaneous amplification and rapid detection of two FVIII intragenic dinucleotide repeats should supersede less informative RFLP analyses.
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06A SINGLE BASE PAIR DELETION IN THE PROMCITER REGION OF THE FACTOR IX FENE IS A S S F I A T E D WITH HAEMOPHlf-lA ,B. . A Hall , A Chuansumnt IR PeAeI. p & !A!kh~~l. Sectlon ot Molecular Geneucs. Department of Medicine
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and Pharmacoloev. Koval Hallamshire Hosoital. Shefield SIO ZJF. bepamncm ot Faedlamcs, R a m a t h b d Hospital. Bangkok, Thaland
A rare class of Christmas disease pauents exemplified by haemophilia B Leyden, suffer From haemophlia B before pubertv but not in later life The mUtahOIlS in a number ot such panenu have now been charactensed and shown to be locahsed to the promoter reFon of the gene encompassmg the s t a n site of transcnpuon at nucleoude + I These stud~es together w t h results obmned trom m mammalian cell culture work have detmed and demonstrated the hmchOrIal importance ot a number of mscnpnon tactor bmdmg sites m thrs region of the gene The region spandmg nucleoudes + 1 to + I 3 is now known to be a bhdmg site tor the transcnpuon tactor ClEBP me hrncuonal is illustrated hv the itlentlficatlon ot significance of ttus site 10
smgle base submtuuons or delehons at posiuons + I . + 6 . + 8 a d + I3 in pauents trom haemophha B Leyden-like pe&grees These pauents have factor LX clottlng levels (F IX C) rangmg trom < I to 3 u/dl before puberty nsmg to o w normal levels in later lite We have chmctensed, by PCR and direct sequeocmg techmques. a previously unreported single nucleoude delehon from a tnplet of t h y m e rcsidues at nucieohdes +6. f 7 and +8 UI the factor LX C E B P bmdmg site from a 6 year old Tha pahent with a F IX C level of 5 ddl Although we have not formally excluded the possihtlitv that there is another mutahon elsewhere in the gene. by inlerence trom the above data we would predict that ttus nucleoude delehon IS responsible tor the haemophha in thu; panent and that the symptoms will gradually ameliorate with age Funchonal chloramphemcol acetyl transfcrase (CAT) assays to compare the mnscnpuonal acuvity of this altered C/EBP bindmg site with its wild-type counterpart are UI progress m order to venfy thu;prediChOfl.
OSSEVEAE HAEMOPHILIA B CAUSED BY A NON-CONSERVATIVE AMINO ACID SUBSTITUTION IN THE SIGNAL PEPTIDE OF FACTOR IX Green PM', Mitchell VE2, Goldman E', Giannelli F' 'Division of Medical 8 Molecular Genetics, UMDS. Guy's Hospital London 'Department of Haematology, Leicester Royal Infirmary, 3HaemophiliaCentre, Royal Free Hospital London
O ~ I X R O M B I NGENERATION IN VIVO PROMOTES THE SELECTIVE LOSS OP HIGH MOLECULAR WEIGHT MIJLTIMERS (HMWM) OP VON WILLEBRAND PACTOR (vWP) AND THE REVERSIBLE SEQUESTRATION OP PLATELETS. C.H. T o h . A . R . Giles". Royal H a l l a m s h i r e H o s p i t a l , Sheffield. UK. and "Queen's University, Kingston. Canada.
Our work on the characterisation of the factor IX mutations in the UK and work from other laboratories throughout the world has demonstrated the extreme mutational heterogeneity of haemophilia B One hundred and forty one different amino acid substitutions have been reported so far and these affect all domains of the factor IX protein except the prepeptide We report the first patient with a mutation in this region that is responsible for the transport of the protein through the endoplasmic reticulum and, hence, for its tl A) in the first exon of secretion A single base substitution ( factor IX (nt 79) results in the replacement of isoleucine -30 by asparagine This breaks the hydrophobic core, that is believed to be one of the essential features of prepeptides. by the replacement of isoleucine by the polar asparagine No other mutation has been found in the other essential regions of the factor IX of this patient (promoter, exons and ANA processing signals) and therefore we propose that the severe disease of this patient is due to inactivation of the prepeptide and hence a failure to secrete factor IX in the circulation This proposal is consistent with the lack of detectable factor IX antigen in the patient's blood
phosphatidylserine (PCPS) vesicles into animal models g e n e r a t e s thrombin in vivo and promotes coagulant. anticoagulant and fibrinolytic responses. We have now demonstrated that there 1s an imnediate loss of vWP antigen with a progressive recovery to normal levels by 65 minutes. Qualitatively. the loss is selective and confined to H M . This is not a s a result of proceolysis and the response is dose dependent with regard to the thrombin generating potential of each dose of P.Xa/PCPS. In the chimpanzee, there is a direct dose-dependent relationship but at the highest dose, a reversal of the vWP response occurred with levels increasing to twice that of baseline values by two minutes. The elevatron persisted at 9 0 minutes Postinfusion and multimeric analysis demonstrated this to be in the f o r m o f unusually large vWF multimers whlch had increased ristocetin co-factor specific activity. This phenomenon was also observed when the thrombin generating effect of P.Xa/PCPS was potentiated by the prevention of protein C activation in vivo. These changes in vWP would suggest that the response observed a t the lower doses reflected a balance of consumption over release of v W P and at the hiqhest dose. the latter effect predomrnrted. Similar chanRes were observed in the platelet count but a direct correlation with dosage was seen in all ases with recovery to normal levels by 45 minutes. ''Chromium labelled platelet studies demonstrated that the process involved was temporary sequestration rather than consumption. These studies suggest that a procoagulant stimulus promotes the temporary l o s s of platelets from the circulation apparently linked to changes in HMWU-vWP and that in the normal animal. this change is reversible. This model should provide the means for studying vWP/platelet/ vessel wall Interactions in vivo.
We have previously described that the infusion of activated Pactor X (P.Xa) in combination with phosphatidylcholine/
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British SocieQ for Haemostasis and Thrombosis
08 THE INFLUENCE OF INFUSIOSS O F I-DESAMINO-B-DARClNlNE VASOPRESSIN (DOAVPI IN \'IVO ON THROhlBIN GENERATION IN VITRO SH lhbotson and PJ Grant. Academic Unit of Sleilicine, 'The General Infirmary. Leeds, LSI 3EX High coagulant factor \'Ill (FVIII:CI and von Willebrand factor (VWFI levels have been reported in conditions associated with thrombo-occlusive disease, such as diabetes. It is thought that the elevation of VWF' represents endothelial injury but it is not known whether high lWlIl:C concentraticins contribute directly t o t h e incidence of thrombotic disease or are a secondary phenomenon. The aim of this work was to investigate the influence of increasing FV1II:C in-vivo on procoagulanr changes ex-vivo. This was achieved tJy studving ihe e f f e c t of infusing DOAVP in 10 healthv vnlunteers on rhrcNmbin generation invitro. nlntid samples u w p tiiken a t Intervals Iwfnre. during and a f t e r B 15 min ~iifiision r ~ f DDAVF'. F\'lll:C rose from (median) 0.42 and 0.43 Il)/ml hefore I>I>AVP i n 1.38. 1.73 and 1.78 IL:/ml a t 15. 31) and 611 miri respecri\*elv IpI.L o n d o n .
Haemostasis i s a b n o r m a l i n u r a e m i c p a t i e n t s , who s u f f e r f r o m b o t h a bleeding t e n d e n c y a n d a l s o p a r a d o x i c a l l y f r o m a n i n c r e a s e in c a r d i o v a s c u l a r m o r t a l i t y . We h a v e p r e v i o u s l y d e m o n s t r a t e d t h a t p l a t e l e t s from t h e s e p a t i e n t s s h o w r e d u c e d s e n s i t i v i t y t o b o t h n i t r i c o x i d e ( S O ) a n d sodium n i t r o p r u s s i d e , which a c t v i a s o l u b l e g u a n y l a t e c y c l a s e . b u t normal s e n s i t i v i t y t o p r o s t a c y c l i n . which a c t s v i a a d e n y l a t e cyclase. Ke h a v e now m e a s u r e d cGP1P a n d cA?lP ( b o t h b a s a l a n d s t i m u l a t e d ) I n w a s h e d p l a t e l e t s from Liraemic p a t i e n t s on e i t h e r r e g u l a r h a e r n o d i a l y s i s (HD) or c o n t i n u o u s a m b u l a t o r y p e r i t o n e a l d i a l y s i s [CAPD). a n d from h e a l t h y c o n t r o l s . The a b i l i t y or n i t r i c o x i d e (10. 0.5-20umolil) t o i n h i b i t t h r o m b i n i n d u c e d p l a t e l e t a q g r e g a t i o n w a s r e d u c e d i n HD p a t i e n t s (n=61 compared with h e a l t h y c o n t r o l s i n - b ) , a n d t h i s w a s p a r a l l e l e d by a r e d u c t i o n i n NO-stimulated i n t r a - p l a t e l e t cGMP (p
I LZ! A STRUCTURAL-FUNCTIONAL STUDY OF PLATELET
fLlTHROHBTN CENERATJON: A PIVOTAL EVENT IN THE REGULATION OF HEMOSTASIS. Alan R. Giles. Departments of Pathology 6 Medicine. Queen's University. Kingston. Ontario. Canada.
MEMBRANE GLYCOPROTEIN CD36. JL McGrePor, HE1 Ghissasi. L McGregor. B Catimel. INSERM U331. Institute Pasteur. Faculte de medecine Alexis Carrel. F-69372 Lyon, France.
One o f the rmerging dilemnas in our understanding of thq regular ion ot hemastasis rs the ubiquitous nnture of thrombin's activity. For example. on one hand it appears to be essential for its own generation via activation of the essential cofactors V and VIII and yet its activity is limited by its ability to generate activated prorrin C (APC). Failure in its generation, p p . hemophilia A and B . results in a devastating hemostatic deficit whereas inappropriate generation, eg. in DIC. may be equally devastating and frequently much wrc difficult to manaRe. Although the medical literature is replete with detailed characterizations o f individual aspects of thrombin's activity. there 1s a major nerd to ent.ablish and characterize the mechanisms by which these interacting and divergent processes are regulated in Y I V O . W e now have considerable experience with an animal model that shows promise in asfiistinr: rhis process. A procoagulant stimulus is developed in normal or hemstatlcilly compromised animals by the intusion of two components o f the prothrombinase complex, factor Xa and procoagulant active phospholipid in the form of vesicles comprised o f phosphatidylcholine/phosphat.idvlserine (PCPS). This combination has been shown to induce a positive hemostatic efFect in bypassing factor VIII and correcting the bleeding diathesis in factor VIII deficient animals. However, the response depends on the absolute and relative concentration of each component to the other. Paradoxically. manipulation of this may induce a bleeding diat,hesis in normal animals. We have monitored the effects ~ n d u c e d on a number of hemostatic mechanisms including von Willrbrand factor. protein C and the fibrinolytic system. Substantla1 changes occur in all three systems that would promote both procoagulant and anticoagulant activities. The mechanisms that dlctate as to whether a net negative or porit.ive h m m s t a t i c rrsult ensues remain to be fully elucidated. However, the availability of coagulant active phospholipid emerges as an essential issue in this outcome.
Clycoprotein CD36. also known as GpIllb or CplV. is a major glycoprotein (GP) present on platelets and also on monocytes. melanomas and endothelial cells. CD36 plays an important role in platelets as one of the receptors to thrombospondin (TSP) and collagen. Moreover. recent studies have shown that Plasmodium falcioarum infected erythrocytes bind to purified CD36. The site on CD36 that interacts with TSP. collagen or malaria infected erythrocytes remains to be determined. In this study plots of hydrophilicity. hydrophobicity. alpha-helices. betasheets. segment mobility and acrophilicity were ?erformed on CD36 (Appligene. Strasbourg). Ten 'iydrophylic peptides spanning the entire amino acid sequence. deduced from the cDNA sequences of CD36, were selected in order of decreasing antigenicity and synthesized using Fmoc chemistry on an Applied Biosystems 431A. Three peptides Pa. Pb. Pc were found to bind to purified TSP using an ELISA system. The binding of TSP to peptide Pb. but not Pa, is inhibited by EDTA. Peptide Pa showed 55% less binding to TSP than peptide Pb. Peptide Pa when added at 200pM. prior to stimulation of platelets by thrombin (0.2-0.4Ulml). inhibited respectively aggregation and endogenous TSP binding. These studies indicate that the platelet CD36 molecule may have two different sites involved in interacting with thrombospondin.
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British Society for Haemostasis and Thrombosis
I L ~ C E L L U L A INTERACTIONS R WITH HEPAILIN:NEW PERSPECTIVES ON AN OLD DRUG. ,J P BiPnnon. A Minter, CN Chesterman. Heart Research Institute and Prince of Wales Hospital, Sydney, Australia. Although the anticoagulant activity of heparin is evidenced by its ability to enhance the activity of ATIII and prevent the clotting of plasma, it is becoming increasingly clear not only that a range of cellular elements are crucial to thrombosis but also that heparin is a multifunctional drug. I n this context we are examining its effects on the cells in the vascular tree, and its ability to modulate the synthesis and release of entdothelial products relevant t o coagulation and fibrinolysis. Human umbilical vein endothelial cells (HUVEC) were grown for 6 days with or without heparin and endothelial growth factor (ECGF). These supplements dramatically inhibited both the basal and thrombin challenged release of prostacyclin (PGI2), plasminogen activator inhibitor 1 (PA1- 1) and thrombospondin (TSP), and and stimulated synthesis of of tissuetype plamnincgen activator (t-PIi) and von Willebrand factor (vWF) . Both supp1ement;s were required for some of these responses, consistent with the effect of heparin being mediated through stabilisation or stimulation of the function of ECGF. However, heparin was able to modulate TSP and vWF release in the absence of exogenous ECGS, which implies a direct mechanism of control by heparin. In general these effects of heparin could stimulate platelet activation and adhesion, and promote fibrinolysis. They are also the opposite of 'those reported to be generated by inflammatory mediators such as interleukin 1 (IL-1). Preconditioning of H W E C with heparin and ECGF counteracted the effects of IL-1 on PGI2 and vWF release. In addition we have used a model system in which H W E C are cultured on filters to demonstrate that both unfractionated and LMW heparin reduce the permeabilit!{ of the endothelial monolayer t o plasma proteins. Such activities may contribute to a physiologically relevant antii n f l m t o w activitv.
OT IDENTIFICATION OF ANTITHROMBIN d i N A IN PERIPHERAL BLOOD LYMPHOCYTES AND ITS USE IN THE CHARACTERISATION OF ANTITHROMBIN VARIANTS. P e w DJ. Carrel1 RW. Dcpt. of Haematology. University of Cambndge. MRC Ccnue. Hills Rd.. Cambridge. CB2 2Q.H.
The charactensation of the dysfunctional variants of many proteins has proved: invaluable in identifying key residues involved in heir function. Despite advances in rapid screening techniques, he complexity of many genes makes the idennfication of DNA mutations a daunang task. A more logical approach would be to analyse the mRNA for a specific pirotein and although for many proteins mRNA is limited to specific organs cg Annthrombin (AT) and the liver, there is now increasing evidence that small amounts of a correctly spliced trmscnpt for vanous tissue-specific gerles can be detected i n a wide vanety of non-\pecific cells. We have developd a melhod for detecting AT mRNA in penpherd blood lymphocytes and show its value in the idennfication 01 AT v;inmts. Total cellular RNA was isolated from penphen.1 blood lymphocytes. A senes of nested oligonucleotide pnmers were designed to amplifv the entre AT cDNA in two overlapping fragments of X03bp :md 760bp: The extreme downstream oligonucleotides were used as pnniers for the reverse uanscnption. Following reverse aanscnption two separate PCR amplifications were pertomed using the nested pnmer!;. After the first PCR, no bands were seen but atter the second PCR. fragments of the expected sizes were clearly visible. Rcstnction mapping of these 2 fragments confumed them to be pans of the AT cDNA. More recently we have shown that it is possible to obtain a full length AT cDNA by modifying the PCR parameters and using the extreme 5' md 3' pnmers in the amplification reaciions.
In order to evaluate this approach i n the identification of AT vmants. total RNA was isolated from a dysfunctional vanant (AT Brighton. 281 lie-Am). reverse uanscnbed and amplified. The product was punfied and directly sequenced. A single additional band was observed corresponding to a T-A subsnturion and confirming the mutation previously identified at the genomic level tDaly et al: unpublished). We have demonstrated that AT mRNA is present in penpheral blood lymphocytes. that i t can be readily amplified. This approach provides a simple and rapid method for the detection of mutations within the AT gene and should prove useful i n the charactensarion o f diverse genetic diborders.
02A RECURRENT 6 BASE PAIR DELETION IN THE ANTITHROMBIN GENE IDENTIFIED BY DNA HETERODUPLEX DETECTION B.J. Ol&. D. A. Lane. C. H. Beresford. U. Abildgaard. S . L. Thein. Institute of Molecular Medicine, John Radcliffe Hospital. Oxford; Depanment of Haematology, Channg Cross and Westminster Hospital Medical School, London; Depanmenr of Pathology, University of Otago Medical School. Dunedin, New Zealand; Depanment of Medicine, Aker Hospital. Oslo. Norway.
In 'ype la antithrombin deficiency. i n which plasma levels of the inhibitor measured by immunological assays approximate 50% of normal, the abnonnality in the majority of individuals appears to be a null allele resulting from a point mutation. As p a n of the stategy for the investigation of the genomic basis of these deficiencies we screened for the presence of DNA hcteroduplexes i n amplified brgments of the gene spanning the antithrombin protein coding regions. Hetercduplexes with a similar pattern of altered mobility were identified i n two families with type la antithrombin deficiency, in the region of exon 3A. The amplified products were directly sequenced in each of the kindreds and sequence analysis showed identical 6 base pair deletions within this region; haplotype analysis of the mutant alleles confinned that the deletions had probably arised independently i n the two families. The deletion occurred within the sequence TIT AAG ? T T (codons 106-lox). removing either TTT AAG or AAG TTT.The deletion site is clearly tlanked by a repeat sequence (TIT.and the complete removal of one repeat and the intervening bases (AAG) is compatible with slippage and mispairing at the repeat site dunng DNA replication as a mechanism for the deletion. Although the mutation removes two intact codons and does not result in a frame shift in trmslation a mutant protein could not be detected in the plasma. It may be that the predicted variant protein is highly unstable. as the deleted bases encode residues which form the turn at the end of helix C in the tertiary structure models of the serpins. and the hydrophobic F108 is predicted to lie in the inienor of the protein.
03 INHERITED ATIII
DYSFUNCTION CAUSED BY THE INTENSIVE EXPRESSION OF AN OTHERWISE NORMAL PLASMA COMPONENT VS Lindo, E M e l i s s a r i , M Learmonth, DN Cooper, W Kakkar. Thrombosis Research I n s t i t u t e , Emmanuel Kaye Building, Manresa Road, London SW3 6LR.
A f a m i l i a l , f u n c t i o n a l ATIII d e f e c t was s t u d i e d i n t h r e e members of a l a r g e family who s t a r t e d s u f f e r i n g severe venous thromboembolism a f t e r the age of 18. Both anti-Xa and antithrombin heparin cofactor a c t i v i t i e s were decreased a t 60% of normal, whereas progressive ATIII a c t i v i t y was found t o be reduced a t 4 5 % . I n c o n t r a s t , ATIII a n t i g e n l e v e l s were normal. Crossed immunoelectrophoresis of p a t i e n t s plasma a g a i n s t antiserum t o human ATIII, i n t h e presence and absence of heparin, was no d i f f e r e n t t o t h a t of t h e c o n t r o l . ATIII p u r i f i c a t i o n by dextran s u l p h a t e p r e c i p i t a t i o n followed by heparin seeharose a f f i n i t y chromatography and e l u t i o n by NaCl g r a d i e n t (0.15-2.OM), showed a normal p r o t e i n recovery and e l u t i o n P r o f i l e , i n a d d i t i o n t h e p u r i f l e d ATIII f r a c t i o n s showed normal progressive a c t i v i t y , heparin c o f a c t o r and anti-Xa a c t i v i t i e s . The p a t i e n t s ' p u r i f i e d ATIII a l s o had a normal mobility on SDS-polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) and formea an SDS s t a b l e complex with p u r i f i e d human thrombin. In a d d i t i o n , CNBr cleavage of t h e p u r i f i e d p r o t e i n followed by HPLC a n a l y s i s and SDS-PAGE d i d not show any abnormal fragments. PAGE, however, of p a t i e n t s plasma on 7 . 4 % n a t i v e g e l s , immunoblot and probing with antiserum t o human ATIII followed by enhanced chemiluminescence ( E C L ) d e t e c t i o n , showed t h e presence o f an i n t e n s e l y immunoreactive band which was not seen i n t h e p u r i f i e d ATIII o r i n p a t i e n t s ' plasma i n t h e presence of SDS. Ferguson p l o t a n a l y s i s showed t h e band t o have a .W of approximately 1 9 4 , 0 0 0 Da. Inununob l o t and ECL a n a l y s i s of 1 2 o t h e r p a t i e n t s ' plasma ( A T 1 1 1 a n t i g e n s 110-160%) d i d not show t h i s i n t e n s e band. Further i n v e s t i g a t i o n s revealed t h a t t h e ATIII a c t i v i t y recovered only when t h e band was removed through t h e p u r i f i c a t i o n Process although no loss of ATIII antigen had occurred. We t h e r e f o r e conclude t h a t t h i s i n h e r i t e d ATIII dysfunction i s caused by t h e i n t e n s i v e expression of an otherwise "normal" plasma component a c t i n g a s an " i n h i b i t o r " , because i t s removal through t h e p u r i f i c a t i o n process r e s u l t s i n 100% recovery of f u n c t i o n a l a c t i v i t y of ATIII and because gene analvsls has Shown no abnormalities i n the ATIIL a ~ u e
Abstracts 04
FIAEhIOPHILIA A DIAGNOSIS B Y ANALYSIS OF A NOVEL DINUCLEOTIDE TANDEM REPEAT SEQUENCE WITHIN THE FACTOR VllIGENE. M.R.A. Lalloz, J . H . McVey, K . MichdeLides and E . G . D . Tiiddenham. Haemostasis Research Group, Clinical Researvh Centre, Harrow, Middx. H A 1 3UJ, U . K . The factor VlIl (FVIII) gene comprises 26 exons spanning 186Kb of DNA located a t t h e distal e n d of the long arm of the Xchromosome. Defects in this gene cause haemophilia A , a bleeding disorder affecting 1:5000 males. Restriction fragment length polymorphisms ( RFLPs) used in c a r r i e r detection a n d prenatal diagnosis a r e informative in only 75% of cases. We recently reported a highly informative ( > g o % ) multi-allelic dinucleotide tandem repeat. (CA),, specific to intron 13of t h e gene (Lallozet a1 1991. Lancet 3 3 8 , 207-11). We have identified and characteriseda new dinucleotide repeat of the form (CA),(TC),, locahsed b y wnventional mapping to intron 22 of the gene. Primers to IINA sequence flanking the intron 22(CA).,(TC).. repeat were i!hosen to amplify genormc DNA samples using conditions for polymerase chain reaction ( P C R ) similar to those used f(rr the intron 13(CA), r e p e a t . Primer specificity was demonstrated by t h e presence of bands corresponding to intron 13( C A ) , . and 2 2 ( CA),( TC).. repeats a f t e r simultaneous PCR of normal genonuc D N A but absence of the latter band only when patient DNA with a deletion mutation s p a n m n g exons 14 to 26 of t h e FVIII gene w a s amplified u n d e r identical conditions. Two intron 22(CA).,(TC)..allelic b a n d s were observed. one comprising 2 5 , the other 26 dinucleotide repeating units. Family studies showed Mendelian inheritance with allelic frequencies ( 5 5 Xchromosomes analysed) of 33% for t h e s h o r t e r and 67% for t h e longer repeat. The iritron 22(CA)..(TC)., repeat tracks with haemophlia A in family studies. The c a r r i e r s t a t u s of two s i s t e r s previously indistinguishable because of identical haplotypes is resolved (one normal the other a n informative c a r r i e r ) following analysis of t h e intron 2 2 repeat. The occurrence of a spontaneous mutation in a three grneration family s t u d y is confirmed to be grandpaternal in origm a f t e r analysis of this r e p e a t . Similar analysis of a chorionic villus sample revealed a n unaffected male fetus to be carried b y a female otherwise uninformative f o r all intragenic FVIII markers analysed . Simultaneous amplification and rapid detection of two FVIII intragenic dinucleotide repeats should supersede less informative RFLP analyses.
3
06A SINGLE BASE PAIR DELETION IN THE PROMCITER REGION OF THE FACTOR IX FENE IS A S S F I A T E D WITH HAEMOPHlf-lA ,B. . A Hall , A Chuansumnt IR PeAeI. p & !A!kh~~l. Sectlon ot Molecular Geneucs. Department of Medicine
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and Pharmacoloev. Koval Hallamshire Hosoital. Shefield SIO ZJF. bepamncm ot Faedlamcs, R a m a t h b d Hospital. Bangkok, Thaland
A rare class of Christmas disease pauents exemplified by haemophilia B Leyden, suffer From haemophlia B before pubertv but not in later life The mUtahOIlS in a number ot such panenu have now been charactensed and shown to be locahsed to the promoter reFon of the gene encompassmg the s t a n site of transcnpuon at nucleoude + I These stud~es together w t h results obmned trom m mammalian cell culture work have detmed and demonstrated the hmchOrIal importance ot a number of mscnpnon tactor bmdmg sites m thrs region of the gene The region spandmg nucleoudes + 1 to + I 3 is now known to be a bhdmg site tor the transcnpuon tactor ClEBP me hrncuonal is illustrated hv the itlentlficatlon ot significance of ttus site 10
smgle base submtuuons or delehons at posiuons + I . + 6 . + 8 a d + I3 in pauents trom haemophha B Leyden-like pe&grees These pauents have factor LX clottlng levels (F IX C) rangmg trom < I to 3 u/dl before puberty nsmg to o w normal levels in later lite We have chmctensed, by PCR and direct sequeocmg techmques. a previously unreported single nucleoude delehon from a tnplet of t h y m e rcsidues at nucieohdes +6. f 7 and +8 UI the factor LX C E B P bmdmg site from a 6 year old Tha pahent with a F IX C level of 5 ddl Although we have not formally excluded the possihtlitv that there is another mutahon elsewhere in the gene. by inlerence trom the above data we would predict that ttus nucleoude delehon IS responsible tor the haemophha in thu; panent and that the symptoms will gradually ameliorate with age Funchonal chloramphemcol acetyl transfcrase (CAT) assays to compare the mnscnpuonal acuvity of this altered C/EBP bindmg site with its wild-type counterpart are UI progress m order to venfy thu;prediChOfl.
OSSEVEAE HAEMOPHILIA B CAUSED BY A NON-CONSERVATIVE AMINO ACID SUBSTITUTION IN THE SIGNAL PEPTIDE OF FACTOR IX Green PM', Mitchell VE2, Goldman E', Giannelli F' 'Division of Medical 8 Molecular Genetics, UMDS. Guy's Hospital London 'Department of Haematology, Leicester Royal Infirmary, 3HaemophiliaCentre, Royal Free Hospital London
O ~ I X R O M B I NGENERATION IN VIVO PROMOTES THE SELECTIVE LOSS OP HIGH MOLECULAR WEIGHT MIJLTIMERS (HMWM) OP VON WILLEBRAND PACTOR (vWP) AND THE REVERSIBLE SEQUESTRATION OP PLATELETS. C.H. T o h . A . R . Giles". Royal H a l l a m s h i r e H o s p i t a l , Sheffield. UK. and "Queen's University, Kingston. Canada.
Our work on the characterisation of the factor IX mutations in the UK and work from other laboratories throughout the world has demonstrated the extreme mutational heterogeneity of haemophilia B One hundred and forty one different amino acid substitutions have been reported so far and these affect all domains of the factor IX protein except the prepeptide We report the first patient with a mutation in this region that is responsible for the transport of the protein through the endoplasmic reticulum and, hence, for its tl A) in the first exon of secretion A single base substitution ( factor IX (nt 79) results in the replacement of isoleucine -30 by asparagine This breaks the hydrophobic core, that is believed to be one of the essential features of prepeptides. by the replacement of isoleucine by the polar asparagine No other mutation has been found in the other essential regions of the factor IX of this patient (promoter, exons and ANA processing signals) and therefore we propose that the severe disease of this patient is due to inactivation of the prepeptide and hence a failure to secrete factor IX in the circulation This proposal is consistent with the lack of detectable factor IX antigen in the patient's blood
phosphatidylserine (PCPS) vesicles into animal models g e n e r a t e s thrombin in vivo and promotes coagulant. anticoagulant and fibrinolytic responses. We have now demonstrated that there 1s an imnediate loss of vWP antigen with a progressive recovery to normal levels by 65 minutes. Qualitatively. the loss is selective and confined to H M . This is not a s a result of proceolysis and the response is dose dependent with regard to the thrombin generating potential of each dose of P.Xa/PCPS. In the chimpanzee, there is a direct dose-dependent relationship but at the highest dose, a reversal of the vWP response occurred with levels increasing to twice that of baseline values by two minutes. The elevatron persisted at 9 0 minutes Postinfusion and multimeric analysis demonstrated this to be in the f o r m o f unusually large vWF multimers whlch had increased ristocetin co-factor specific activity. This phenomenon was also observed when the thrombin generating effect of P.Xa/PCPS was potentiated by the prevention of protein C activation in vivo. These changes in vWP would suggest that the response observed a t the lower doses reflected a balance of consumption over release of v W P and at the hiqhest dose. the latter effect predomrnrted. Similar chanRes were observed in the platelet count but a direct correlation with dosage was seen in all ases with recovery to normal levels by 45 minutes. ''Chromium labelled platelet studies demonstrated that the process involved was temporary sequestration rather than consumption. These studies suggest that a procoagulant stimulus promotes the temporary l o s s of platelets from the circulation apparently linked to changes in HMWU-vWP and that in the normal animal. this change is reversible. This model should provide the means for studying vWP/platelet/ vessel wall Interactions in vivo.
We have previously described that the infusion of activated Pactor X (P.Xa) in combination with phosphatidylcholine/
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British SocieQ for Haemostasis and Thrombosis
08 THE INFLUENCE OF INFUSIOSS O F I-DESAMINO-B-DARClNlNE VASOPRESSIN (DOAVPI IN \'IVO ON THROhlBIN GENERATION IN VITRO SH lhbotson and PJ Grant. Academic Unit of Sleilicine, 'The General Infirmary. Leeds, LSI 3EX High coagulant factor \'Ill (FVIII:CI and von Willebrand factor (VWFI levels have been reported in conditions associated with thrombo-occlusive disease, such as diabetes. It is thought that the elevation of VWF' represents endothelial injury but it is not known whether high lWlIl:C concentraticins contribute directly t o t h e incidence of thrombotic disease or are a secondary phenomenon. The aim of this work was to investigate the influence of increasing FV1II:C in-vivo on procoagulanr changes ex-vivo. This was achieved tJy studving ihe e f f e c t of infusing DOAVP in 10 healthv vnlunteers on rhrcNmbin generation invitro. nlntid samples u w p tiiken a t Intervals Iwfnre. during and a f t e r B 15 min ~iifiision r ~ f DDAVF'. F\'lll:C rose from (median) 0.42 and 0.43 Il)/ml hefore I>I>AVP i n 1.38. 1.73 and 1.78 IL:/ml a t 15. 31) and 611 miri respecri\*elv IpI.L o n d o n .
Haemostasis i s a b n o r m a l i n u r a e m i c p a t i e n t s , who s u f f e r f r o m b o t h a bleeding t e n d e n c y a n d a l s o p a r a d o x i c a l l y f r o m a n i n c r e a s e in c a r d i o v a s c u l a r m o r t a l i t y . We h a v e p r e v i o u s l y d e m o n s t r a t e d t h a t p l a t e l e t s from t h e s e p a t i e n t s s h o w r e d u c e d s e n s i t i v i t y t o b o t h n i t r i c o x i d e ( S O ) a n d sodium n i t r o p r u s s i d e , which a c t v i a s o l u b l e g u a n y l a t e c y c l a s e . b u t normal s e n s i t i v i t y t o p r o s t a c y c l i n . which a c t s v i a a d e n y l a t e cyclase. Ke h a v e now m e a s u r e d cGP1P a n d cA?lP ( b o t h b a s a l a n d s t i m u l a t e d ) I n w a s h e d p l a t e l e t s from Liraemic p a t i e n t s on e i t h e r r e g u l a r h a e r n o d i a l y s i s (HD) or c o n t i n u o u s a m b u l a t o r y p e r i t o n e a l d i a l y s i s [CAPD). a n d from h e a l t h y c o n t r o l s . The a b i l i t y or n i t r i c o x i d e (10. 0.5-20umolil) t o i n h i b i t t h r o m b i n i n d u c e d p l a t e l e t a q g r e g a t i o n w a s r e d u c e d i n HD p a t i e n t s (n=61 compared with h e a l t h y c o n t r o l s i n - b ) , a n d t h i s w a s p a r a l l e l e d by a r e d u c t i o n i n NO-stimulated i n t r a - p l a t e l e t cGMP (p
