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846 847 HIGH AFFINITY CALCIUM BINDING TO DOMAINES OF PROTEIN C THAT ARE HOMOLOGUS TO THE EPIDERMAL GROWTH FACTOR M Ohlin and J Stenf!o Department of Cljnical Chemistry University of Lund MalmO General Hospital MalmO. Sweden

In addition to y- (Gia)-dependent A molecular abnormality of protein C (PC) has been found in calcium binding all of the -dependent plasma proteins, an 18-year-old female who has been thrombophilic since she was except prothrombin, have one or two high affinity calcium one-year old. Detailed studies revealed that PC binding sites that do not require the Gla residues. A common antigen was only 25% of normal while PC activity was substantial­ denominator among these proteins (factors IX, X, protein C, ly undetectable in her plasma. Other related plasma proteins and ) is that they have domaines that are including vitamin K-dependent blood coagulation factors, anti­ homologus to the epidermal growth factor (EGF) precursor. In ill and plasminogen were all within normal limits. factors VII,IX,X, protein C and in protein Z the aminoterminal of Family study demonstrated that her father, one of her paternal two EGF homology regions contain one residue of uncles and her half-bred sister had half-normal levels of both ~-hydroxyaspartic acid (Hya) whereas in protein S the activity and antigen of PC. They thus appear to be hetero­ zygotes for PC deficiency. Her mother had a normal level of PC aminoterminal EGF homology region contains Hya and the three as antigen but only 60% of normal as activity. We thus presumed following contain one ~-hydroxyasparagine residue each. that nearly a half population of PC was not properly functioning In an attempt to elucidate the role of the EGF homology regions in her mother's plasma. By utilizing insolubilized monoclonal' in the Gla independent calcium binding we have isolated a tryptic antibodies against PC, we isolated both single-chain and two­ fragment (residue 44-138) from the light chain of human chain forms of PC from the propositus' plasma. They were found protein C. The fragment was isolated using a monoclonal antibody to have normal molecular weights by SDS-PAGE, but they failed to that recognizes a calcium ion stabilized epitope that is expressed exhibit amidolytic activity and to incorporate 3 H-labeled DFP both in intact protein C and in protein C lacking the Gla after thrombin-treatment. By immuno~lotting and ELISA utilizing domaine.The antibody bound the isolated EGF homology region in a monoclonal antibody that recognizes the activation peptide part of the heavy chain of PC, we found that cleavage of the activa­ the presence of calcium ions but not in EDTA containing buffer. A tion peptide by thrombin was impaired totally in the propositus' calcium ion titration showed half maximal binding at and partly in her mother's PC. It is thus most probable that the approximately 200 11M Ca2 +. The metal ion induced propositus inherited PC deficiency from her father and a molecu­ conformational change in the isolated fragment was also studied lar abnormality of non-activatable protein C from her mother, with affinity purified rabbit antibodies against Gla domainless respectively. Accordingly, all the PC molecules in the propositus' protein C. Antibodies that bound in the presence of calcium ions plasma are non-functional, which may have culminated in the and that could be eluted with EDTA recognized the metal ion occurrence of cerebral thrombosis at the age of l and severe induced conformational change in the isolated EGF homology deep venous thrombosis and bilateral pulmonary embolisms at the domain. Our results suggest that one or both of the EGF homology age of l 0. regions are involved in the Gla-independent high affinity calcium binding in the vitamin K-dependent plasma proteins.

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INHIBITION OF HUMAN PROTEIN C ACTIVATION BY VITAMIN K-DEPENDENT The Effect of Changes in the Leader Sequence of Human Protein C on PROTEINS, INVOLVEMENT OF THE y -CARBOXYGLUTAMIC ACID DOMAIN IN Biosynthetic Processing and Gamma-. DISTINCT INTERACTIONS WITH THE HUMAN THROMBIN-THROMBOMODULIN COMPLEX AND PHOSPHOLIPIDS. J .-M. Freyssinet, A. Beretz, C. Klein-Soyer, J. Cauchy, s. Schuhler and J.-P. Cazenave. INSERM U. 311 , Centre R€gional de Transfusion Sanguine, Strasbourg, France.

Protein C (PC) activation by thrombin (T) occurs at the Protein C is the precursor to a serine protease in plasma which endothelial cell (EC) surface in the presence of thrombomodulin contains gamma-carboxy glutamic acid and functions as a potent (TM). Reconstitution of purified TM into phospholipid (PL) . Protein C shows considerable structural homology with vesicles results in an increased activation of PC but not of Gla­ the other vitamin K-dependent coagulation factors including domainless-PC ( GD-PC), a chymotryptic derivative of PC lacking the prothrombin, factor VII, factor IX and . This homology y-carboxyglutamic acid domain (Gla-domain). We show that several includes the putative pro-peptide region of the prepro leader sequences human vitamin K-dependent proteins can interfere in the activation for these proteins, as well as the leader sequences for of human PC by the human T-TM complex either in the presence or in gamma-carboxylated proteins from bone. Deletion mutants have been the absence of PL. Prothrombin fragment 1 (F1), peptide 1-41, the constructed in the eDNA for human protein C in order to test the

N-terminal chymotryptic Gla-domain of prothrombin (F II), FII and possibility that the pro-peptide portion of the 42 amino acid leader This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. factor X (FX) were able to inhibit PC activation, They had no effect sequence serves as a molecular signal for gamma-carboxylation. on the amidolytic activity of activated PC. Non-competitive Accordingly, these mutants contain the pre-peptide (hydrophobic inhibition was observed in the presence of 10 ~M F1 when PC, at leader) plus portions of the pro-peptide at the amino terminus of the various concentrations, was activated by the 8 nM T-TM complex, at light chain. The mutant proteins were expressed in carboxy Ia !ion­ 2 mM ca2+, with or without PL-reconstituted TM. In any case the competent mammalian cells and analyzed by barium citrate apparent Km remained unchanged at 2 1M. In the presence of precipitation and N-terminal amino acid sequencing. These studies optimal PL concentrations and in the absence of F1 , the Vmax could ·have shown that deletions in the pro-peptide region interfere with be enhanced up to 9-fold. When F1 was present, the extents of gamma-carboxylation and removal of the pro-peptide. Deletion of inhibition with and without PL were comparable and resulted in a~­ residues -1 through -12 had little effect on the carboxylation or fold decrease of the Vmax. These effects were independent of Ca + secretion. Deletion of -1 through -17 completely abolished gamma­ between 1 and 5 mM and ofT between 10 and 50 nM. At 0.6 ~M PC, half carboxylation, but had no measurable effect on secretion. Amino maximal inhibition occurred at 8 ~M F1 and 1 )JM peptide 1-41 in the terminal sequence analysis of the latter mutant showed that the light presence or in the absence of PL. ProteinS and factors VII and IX chain began with Thr-Pro-Ala-Pro... This corresponds to a sequence in had only minimal effect. The inhibition due to F1 and FX was also the prepro leader starting at -24. This indicates that the signal noticed when PC was activated by T in the presence of cultured human peptidase cleavage site for human protein C is between residues -25 vascular EC. A Ki of 4 ~M could be determined for F1 with EC-bound and -24 and removal of the pro-peptide had been blocked by the TM. The non-competitive character was confirmed by the observation deletion. that F1 could also inhibit the activation of GD-PC by the T-TM complex. Incomplete heat-decarboxylation of F1 and FII, partially abolished their capacity to inhibit PC activation. These results suggest that the Gla domain of PC is involved in two distinct types of interactions. This vitamin K-dependent functional entity is necessary to allow the enhancement of PC activation by anionic PL and also interacts with the T-TM complex.

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