NOVEL PROCOAGULANT PROTEINS

Abstract: Novel procoagulant proteins which comprise the amino acid sequence: A-X-B wherein region A represents the polypeptide sequence Ala-20 through Arg-759 substantially as shown in Table 1; region B represents the polypeptide sequence Ser-1709 through Tyr-2351 substantially as shown in Table 1; and region X represents a polypeptide sequence comprising up to 949 amino acids substantially duplicative of sequences of amino acids within the sequence Ser-760 through Arg-1708 of Table 1, wherein the amino terminus of X is covalently bonded through a peptide bond designated "-" to the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B. Methods of making such proteins and their use in pharmaceutical preparations is also disclosed.

Description: I NOVEL PROCOAGULANT PROTEINS This invention relates to a novel series of proteins which exhibit procoagulant properties. These proteins have marked structural differences from human factor VIII:C, but have 5 similar procoagulant activity. Factor VIII:C is the blood plasma protein that is defective or absent in Hemophilia A disease. This disease is a hereditary bleeding disorder affecting approximately one in 20,000 males. lOThe structure of factor VIII:C is described in U.S. Patent Appli¬ cations Serial No. 546,650 filed October 28, 1983 and No. 644,036 filed August 24, 1984, which are incorporated herein by reference and in Nature. 312:306, 307, 326 and 342. 15 θne of the problems presently encountered with the use of human factor VIII:C for treatment of hemophilia arises from its anti- geniσity. A significant percentage of hemophiliacs have developed an immune reaction to the factor VIII:C used for their treatment. Non-hemophiliacs can also develop or acquire 2 ohemophilia when their immune systems become sensitized to factor VIII:C and produce circulating antibodies or "inhibitors" to factor VIII:C. In either case, the effect is the neutrali¬ zation of whatever factor VIII:C is present in the patient, making treatment very difficult. Until now, the method of 25choice for treating hemophiliacs with this problem has been to administer, in cases of severe bleeding episodes, non-human factor VIII:C, such as treated porcine factor VIII:C. See ernoff et al.. Blood 63:31 (1984). However, the antibodies which neutralize the clotting ability of human factor VIII:C 30will react to a varying extent with factor VIII:C of other species, and the porcine protein is itself antigenic, thus both the short-term and long-term effectiveness of such treat¬ ment will vary. Additionally, patients frequently display adverse reactions to infusion with the porcine factor VIII:C. The use of porcine factor VIII:C in spite of the risks has been justified because of the lack of reliably effective alternatives. Kernoff, supra at 38. The present invention provides an alternative to the administration of porcine factor VIII:C. This invention provides for proteins which have procoagulant activity similar to that of factor VIII:C and also have substant- ially lower molecular weight. These proteins are schematically depicted by formula (1) as follows: (1) A-X-B wherein A represents a polypeptide sequence substantially dupli¬ cative of the sequence Ala-20 through Arg-759; B represents a polypeptide sequence substantially duplicative of the sequence Ser-1709 through the C-terminal Tyr-2351; ahd X represents a polypeptide sequence of up to 949 amino acids substantially duplicative of sequences of amino acids within the sequence Ser-760 through Arg-1708. The amino terminus of region X is covalently bonded through a peptide bond (designated "-" in formula 1) to the carboxy terminus of A. The carboxy terminus of region X is likewise bonded to the amino terminus of B. Numbering of amino acids throughout this disclosure is with reference to the numbering of amino acids in Table 1 in which the first amino acid. Met, of the leader sequence is assigned Number 1. Protein domain X may comprise a continuous but shorter sequence selected from the region Ser-760 through Arg-1708. Alternatively X may comprise two or more amino acid sequences selected from that region which are covalently bonded by a peptide bond (maintaining an ascending numerical order of amino acids) . By way of example, one compound of this invention contains a region X comprising the amino acid sequence of Ser-760 to Pro- TABLE 1 5' GAATTCCCCACTCGGTAACTTCCTTAAATCCTCTGGAAACAA^TTCCGACTTTTCATTAAATCACAAATT TTACT111rTCCCCTCCTGCCACCTAAACATATTTTAGACAAGAATTA-CCrrTTCCTTCTCCACTTCAACATTTCTAGCAA ACTC HET Cln Il« Clu Ltu Sβr Thr Cys Phβ Phβ Leu Cys Leu Leu Arg Phβ Cys Phβ 18 ATC CAA ATA CAU CTC TCC ACC TCC TTC TTT CTC TCC % CTT TTC CCA TTC TCC TTT Sβr Ala Thr Arg Λrg Tyr Tyr L«u Cly Ala Val Clu Lcit Sor Trp Asp Tyr MET 36 ACT CCC ACC ACΛ ΛCA TΛC TAC CTC CCT CCA CTC CAΛ CTC TCΛ TCC CΛC TAT ATC Cln Ser Asp Leu Cly Cl. Leu Pro Val Asp Ala Arg Phβ Pro Pro Arg Val Pro 5. CAA ACT GAT CTC CCT CAC CTC CCT CTC CAC CCA ACA TTT CCT CCT ΛCA CTC CCA Lys Sβr Phβ Pro Phβ A*n Thr Sβr Val Val Iyr Lye Lys Thr Leu Phβ Val Clu 72 AAA TCT TTT CCA TTC AAC ACC TCA CTC CTC TAC AAA AAC ACT CTC TTT CTA CAA Phe Thr Val Hia Leu Phβ Asn lie Ala Lys Pro Arg Pro Pro Trp MET Cly Leu 90 TTC ACC CTT CAC CTT TTC AAC ATC CCT AAC CCA ACC CCA CCC TCC ATC CCT CTC Leu Cly Pro Thr He Cln Ala Clu Val Tyr Asp Thr Val Val lie Thr Leu Lys 103 CTA CCT CCT ACC ATC CAC CCT CAC CTT TAT CAT ACA CTC CTC ATT ACA CTT AAC - Am MET Al* Sβr His Pro Val Sβr Leu Hi* Ala Val Cly Val Ser Tyr Trp Lys 12δ AAC ATC CCT TCC CAT CCT CTC ACT CTT CAT CCT GTT CCT CTA TCC TAC TGG AAA Al* Sβr Clu Cly Ala Clu Tyr Asp Asp Cln Thr Ser Cln Arg Clu Lys Clu Asp *** CCT TCT CAC CCA CCT CAA TAT CAT CAT CAC ACC ACT CAA ACC CAC AAA CAA CAT Asp Lys Vai Phβ Pro Gly Cly Sβr His Thr lyr Val Tcp Cln Val Leu Lys Clu 162 CAT AAA CTC TTC CCT CCT CCA ACC CAT ACA TAT CTC TCC CAC CTC CTC AAA CAC Aβn Cly Pro MET Ala Sβr Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu S*r His 180 AAT CCT CCA ATG CCC TCT CAC CCA CTC TCC CTT ACC TAC TCA TAT CTT TCT CAT Val Asp Leu Val Lys Asp Leu Asn Sβr Cly Leu lie Gly Ala Leu Leu Val Cys 193 CTC CAC CTC CTA AAA CAC TTC AAT TCA CCC CTC ATT GCA CCC CTA CTA CTA TCT Arg Clu Cly Sβr Leu Ale Lys Clu Lys Thr Gin Thr Leu His Lys Phβ Tie Leu 216 ACA CAA CCC ACT CTC CCC AAC CAA AAC ACA CAC ACC TTC CAC AAA TTT ATA CTA Leu Phβ Ala Val Phe Asp Clu Cly Lys Sβr Trp His Ser Clu Thr Lys Asn Ser 234 CTT TTT CCT CTA TTT CAT CAA CCC AAA ACT TCC CAC TCA CAA ACA AAC AAC TCC Leu MET Cln Asp Arg Asp Ala Ala Ser Ala rg Ala Trp Pro Lys MET His Thr 252 TTC ATC CAC CAT ACC CAT CCT CCA TCT CCT CCC CCC TCC CCT AAA ATC CAC ACA Val Asn Cly Tyr Val Asn Arg Ser L«u Pro Cly Leu lie Cly Cys His Arg Lys 270 CTC AAT CCT TAT CTA AAC ACC TCT CTC CCA CCT CTC ATT CCA TCC CAC ACC AAA Sor Val Tyr Trp His Val II* Cly MET Cly Thr Thr Pro Clu Val His Ser lie 288 TCA CTC TAT TCC CAT CTC ATT CCA ATC GGC ACC ACT CCT CAA GTC CAC TCA ATA Phβ Luβ Clu Cly His Thr Phβ Leu Val Arg Asn His Arg Cln Ala Ser L*u Clu 306 rrc CTC CAA CCT CAC ACA TTT CTT CTC ACC ΛAC CAT CCC CAC CCC TCC TTU CAA TABLE 1, continued He S e r Pro lie Thr Phβ l.eu Thr Ala Cln Thr Lou Leu HET Aap Leu Cly CU 324 A TC TCC CC A ATA ACT TTC CTT ACT CCT CΛΛ ΛCΛ CTC TTC ATC CΛC CTT GttΛ CA Phβ ' Leu L < su Phβ Cys His He Ser Ser His Gin His Asp Cly MET Clu Ala Tyr 342 TIT CTA CTG TTT TCT CAT ATC TCT TCC CAC CΛΛ CAT GAT CCC ATG CAA CCT TAT Val Lys Val Asp Ser Cys Pro Clu Clu Pro Gin I.cu Arg HET Lya Λsn Asn Clu 360 CTC A AA GTA CAC ACC TCT CCA GAG CAA CCC CΛΛ CTA CCA ATG AAΛ' ΛAT AAT CAA Clu Ala- Clu Asp Tyr Asp Asp Asp Leu Thr Asp S*r Clu MET Asp Val Val Arg 378 CAA GCG CAA GAC TAT CAT CAT CAT CTT ACT CAT TCT CΛΛ ATC GAT CTC CTC ACC Phβ Asp Aap Asp Asn Sβr Pro Ser Phe He Cln He Arg Sβr Val Ala Lys Lys 396 TTT CAT GAT CAC AAC TCT CCT TCC TTT ATC CAA ATT CCC TCA CTT CCC AAC AAC His Pro Lys Thr Trp Val His Tyr He Aln Ala Clu Clu Clu Asp Trp Λsp Tyr 414 CAT CCT AAA ACT TCC GTA CAT TAC ATT CCT CCT GΛA GAC CΛC CAC TCC CAC TAT Ala Pro Leu Val Leu Ala Pro Asp Asp Λrg Sβr Tyr Lys Sβr Cln Tyr Leu Asa 432 CCT CCC TTΛ CTC CTC CCC CCC CAT CAC ACA ACT TAT AAA ACT CAA TAT TTC AAC Asn Cly Pro Gin AΓR He Cly Arg Lyn Tyr Lys Lys V l Arg Phe MKT Ala Tyr 450 ΛAT CCC CCT CAC CCC ΛTT CCT ACG ΛΛ.C TΛC ΛΛΛ ΛΛΛ CTC CCA TTT ΛTC CCA TΛC Thr Asp Clu Thr Fhβ Lys Thr Arg Clu Ala He Cln His Glu Ser Cly Ho Leu 468 ACA CAT GAA ACC TTT AAC ACT CCT CAΛ CCT ATT CΛG CAT CAA TCA CCΛ ATC TTC Cly Pro £eu Leu Tyr Cly Clu Val Cly Asp Thr Leu Leu He He Phe Lys Asn 486 CCA CCT TTA CTT TAT C.C. GΛA CTT CCA CΛC ACΛ CTC TTC ΛTT ATA TIT ΛΛG AAT Gin Ala Sβr Arg Pro Tyr Asn He Tyr Pro His Cly lie Thr Asp Val Arg Pro S04 CAA CCA ACC ACA CCA TAT AAC ATC TAC CCT CAC CGA ATC ACT CAT CTC CCT CCT Leu Tyr Sβr Arg Arg Leu Pro Lys Cly Val Lys His Leu Lys Asp Phβ Pro He 52 2 TTC TAT TCA ACG ACA TTA CCΛ AAA CCT GTA ΛAA CAT TTC AΛC CAT TTT CCA ATT Leu Pro Cly Clu He Phβ Lys Tyr Lys Trp Thr Val Thr Val Glu Aap Cly Pro 540 CTG CCA CCA CAA ATA TTC AAA TAT ΛΛA TCG ACA CTG ACT CTA GAA CAT GCG CCA Thr Lys Sβr Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn HET s58 ACT AAA TCA CAT CCT CCC TCC CTG ACC CCC TAT TΛC TCT ACT TTC CTT AAT ATC Glu Arg Asp Leu Ala Sβr Gly Leu He Cly Pro eu Leu He Cys Tyr Lys Clu 576 GAG ACA CAT CTA CCT TCA CCΛ CTC ΛTT CCC CCT CTC CTC ΛTC TCC TAC ΛΛΛ CΛA Ser Val Asp Cln Arg Cly Asn Cln He MLT Sur Λsp Lys Λrg Asn Val He Leu 594 TCT CTA GAT CΛA ACA CCA ΛΛC CAG ATΛ ΛTC TCA CΛC ΛΛC ΛCC ΛΛT CTC ΛTC CTG Ph β S β r Vnl P e Asp Cln Λsn Λrg Sor Trp Tyr Leu Thr Glu Asn lie Cln Λrg g 2 rrr TCT CTA TTT CAT CAC ΛΛC CCΛ ΛOC ICC TAC CTC ΛCA CΛG ΛΛT ΛΓA CΛΛ CCC Ph β Leu Pro Asn Pro Ala Cly Val Cln Leu Clu Λsp ro Glu Plic Gin Λla Ser 63 0 TTT CTC CCC AAT CCΛ CCT CCA CTG CAC CTT CAC CAT CCA CAC TTC CΛΛ CCC TCC A sn He HET His Ser 11* Asn Cly Tyr Val Phβ A6p Ser Leu Gin Leu S«r Vβl 648 A AC ATC ATC CAC ACC ATC AΛT CCC 7ΛT CTT TTT CAT ACT TTC CAC TTβ TCA CTT TABLE 1, continued Cys Leu His Clu Val Ala Tyr Trp Tyr lie Leu Sur lie Cly Λla ( .'In Thr Λsp 666 TCT TTC CAT CAG CTG CCΛ TΛC TCC TΛC ΛTT CTΛ ΛCC ΛTT CGΛ CCA CΛG ΛCT CAC Pliβ . Leu Ser Val Phβ Phe Ser Gly' Tyr Thr Phe Lys His I.ys MET Val Tyr Clu --'' TTC CTT TCT GTC TTC TTC TCT CCA TAT ACC TTC AAA CAC ΛΛA ATG' GTC TΛT CAA Asp Thr Leu Thr Leu Phe Pro Phβ Sβr Cly Clu Thr Val Phe MET Ser MET Clu 702 CAC ACA CTC ACC CTA TTC CCA TTC TCA CCA CΛΛ ΛCT CTC TTC ΛTC TCG ATG CAA Aan Pro Cly Leu Trp lie Leu Gly Cys fMβ Asn Ser Λsp Phe Arg Asn Arg Cly 720 AAC CCA CCT CTA TCG ATT CTG GCG TCC CAC AAC TCA CAC TTT CCG AAC ACA CCC MET Thr Ala Leu Leu Lys Val Ser Ser Cys Aap Lye Asn Thr Cly Aap Tyr Tyr 738 ATG ACC CCC TTA CTC AAC GTT TCT ACT TCT GΛC AAC AAC ACT GGT GAT TAT TAC Glu Asp Sβr Tyr Clu Asp lie Ser Ala Tyr Leu eu Sβr Lya Λsn Asn Ala lie 756 CAC GAC ACT TAT CAA CAT ΛIT TCA CCA TAC TTC CTG ACT AAA AAC AAT CCC ATT Clu Pro Arg Ser Phe Ser Gin Aan Sβr Arg ills Pro Ser Thr Arg Gin Lys Gin 774 CAA CCA ACA ACC TTC TCC CAC AAT. TCA AGΛ CAC CCT ΛGC ACT AGO CAA ΛΛC CAA Phβ Aan Ala Thr Thr lie Pro Clu Asn Λsp He Clu Lys Thr Asp Pro Trp Phβ 792 TTT AAT GCC ACC ACA ATT CCA CΛA AAT GAC ΛTΛ CAG AAG ACT CAC CCT TOG TTT Ala His Arg Thr Pro MET Pru Lya He Cln Asn Val Ser Ser Ser Asp Leu Leu 810 GCA CAC AGA ACA CCT ATG CCT AAA ATA CAA ΛΛT CTC TCC TCT ACT CAT TTC TTC MET Leu Leu Arg Gin Ser Pro Thr Pro His Cly Leu Set Leu Ser Λsp Leu Cln 828 ATC CTC TTC CCA CAG ACT CCT ACT CCA CAT CCG CTA TCC TTΛ TCT GΛT CTC CΛA Clu Ala Lys Tyr Clu Thr Phβ Ser Asp Asp Pro Ser Pro Gly Λln Tie Asp Ser 346 GAA GCC AAA TAT CAG ACT TTT TCT CAT GAT CCA TCΛ CCT CCA CCA ΛTΛ CAC ΛCT Asn Asn Sβr Leu Ser Clu MET Thr His Phe Λrg Pro Cln Leu His Ills Ser Gly 864 AAT AAC ACC CTG TCT CΛΛ ΛTC ACA CΛC TTC ΛCC CCA CΛC CTC CAT CΛC ΛCT CCG Asp MET Val Phβ Thr Pro Clu Ser Gly Leu Gin Leu Arg Leu Aβπ Clu Lys Leu 882 CAC ATG CTA TTT ACC CCT GAC TCA CCC CTC CAA TTΛ ΛCΛ TTΛ ΛAT CΛG ΛΛΛ CTC Cly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp The Lys Val Sur Sβr Thr 900 CCG ACA ACT CCA GCΛ ACA GAC TTC AΛG AAA CTT CΛT TTC ΛΛA GTT TCT ACT ACA Ser Asn Aβn Leu lie Ser Thr He Pro Sβr Asp Λsn eu Ma Ala Gly Thr Λsp 918 TCΛ AAT AAT CTG ATT TCA ACA ATT CCA TCΛ GAC AAT TTG GCA CCΛ CCT ACT CΛT Aβn Thr Sβr Ser Leu Cly Pro Fro Ser MET Pro Val His Tyr Λsp Ser Cln Leu 936 AAT ACA ACT TCC TTA CGΛ CCC CCA ACT ΛTC CCA GTT CAT TAT GAT ΛCT CΛA TTA Asp Thr Thr Lαu Phe Cly Lys Lys Ser Ser Pro Leu Thr Clu Ser Cly Cly Tro 954 CAT \CC ΛCT CTA ITT CCC AΛΛ AAG TCA TCT CCC CTT ΛCT CAG TCT GGT CCA CCT Leu Ser Leu Ser Clu Clu Asn Λsn Asp Ser ys Leu Leu Glu Ser Cly Leu MET 9 2 CTG ACC TTG ACT CΛA CΛA ΛΛT ΛAT GAT TCA AA<; TTC TTΛ ι:_A TCΛ CCT TTΛ ΛTC Λsn Ser Gin Glu Sβr Ser Trp Gly Lye Aβπ Val S^r Ser Thr Glu Ser Cly Arg 990 AAT ACC CAA CAA ACT TCA TCG CCA AAA AAT CTA TCG TCA ACA «:AC ACT CGT ACC TABLE 1. continued Leu Phe Lys Gly Lys Λrg Ala His Gly Pro Λla Leu l.eti Thr Lys Λ.;p Λsn Ala I' 008 TTΛ TTT AA A CGG AAA ΛGA CCT CΛT CCΛ CCT CCT TTG TTG ΛCT AAA GAT ΛΛT GCC Leu Phe Lya Val Ser lie Ser Leu Leu Lye Thr Λ&n Lys Thr Ser Λsn Aβn Ser ,02δ TTA TTC AAΛ CTT ACC ATC TCT TTG TTA AΛG ACA AAC ΛAΛ ACT TCC AAT AΛT TCA A la Thr Asn Arg Lye Thr His lie Asp Cly Pro Ser Leu Leu lie Glu Aβn Ser 1,044 CCA ACT AΛT ΛGA - AAC ACT CAC ATT CAT CCC CCΛ TCA TTA TTA ATT GAG ΛAT ACT Pro Ser Val Trp Cln Asn lie Leu Glu Ser Λsp Thr Clu Phe Lys Lys Val Thr 1,062 CCA TCA CTC TCC CAA AAT ATA TTA CAA AGT GAC ACT CAC TTT AAA AAA CTG ΛCA Pro Leu lie His Asp Arg MET Leu MET Asp Lye Asn Λla Thr Ala Leu Arg Le 1 ' 080 CCT TTG ATT CAT GΛC ACA ATG CTT ΛTG CAC ΛΛA ΛAT GCT ACA CCT TTC AGG CTΛ Asn His HET Sβr Asn Lys Thr Thr Ser Ser Lys Λsn MET Glu MET Val Gin Cla 1 ' 098 AΛT CAT ATC TCA AAT AAA ACT ACT TCA TCA AAA ΛCC ATG CAA ATC CTC CAA CAG Lya Lys Glu Gly Pro lie Pro Pro Λsp Ala Gin Λsπ Pro Asp HET Ser Phβ Phe 1,116 AAA AAA CAG GGC CCC ATT CCA CCA CAT GCA CAA AΛT CCA GAT ATC ICG TTC TTT Lys HET Leu P e Leu Pro Clu Ser Ala Arg Tip Tie Gin Arg Thr His Cly Lys 1,134 AAG ATG CTA TTC TTG CCA GΛA TCA CCA ΛCG TCG ATΛ CAΛ AGG ACT CAT CCΛ ΛAG Asn Ser Leu Asn Sβr Cly Cln Gly Pro Sβr Pro Lys Cln Lru Val Ser Leu Gly 1,152 AAC TCT CTC AAC TCT CCG CAA CCC CCC ACT CCΛ AAC CAA TTA GTA TCC TTA GCA Pro Clu Lye Sβr Val Clu Cly Gin Asn Phe Leu Ser Clu Lys Aβn Lys Val Val 1,170 CCA " CAA AAA TCT GTC CAA GGT CAC AAT TTC TTC TCT CΛG AAΛ Λ.C AAΛ CTG CTA Val Cly Lys Cly Clu Flie Thr Lys Λsp Val Gly Leu Lys Glu HET Val Nie Pro 1,138 GTA CCΛ AAC CGT GAA TTT ACA AΛG CΛC GTA CCA CTC AΛA CAG ATC CTT TTT CCA- Ser Ser Arg Asn Leu Phβ Leu Thr Asn Leu Asp Λsn Leu His Glu Λsn Asn Thr 1,206 ACC AGC ACA AAC CTA TTT CTT ACT AAC TTC GAT AΛT TTA CAT CAΛ ΛΛT ΛAT ACA His Asn Gin Glu Lys Lya lie Gin Clu Clu lie Glu Lys Lys Glu Thr Leu ila 1,224 CAC AAT CΛA CAA ΛAA AAA ATT CAC CAA CAΛ ATA CAA ΛAC ΛAG CAA ΛCΛ TTΛ ATC Gin Clu Aβn Val Val Leu Pro Cln lie His Thr Val Thr Gly Thr Lys Λsn Phe 1,242 CAA GAG AAT GTA CTT TTC CCT CAC ATA CAT ACΛ CTC ACT CCC ACT AAG ΛΛT TTC HET Lys Asn Leu Phe Leu Leu Ser Thr Λrg Cln Asn Vnl Clu Gly S«r Tyr Glu 1,260 ATC AAC AAC CTT TTC TAA CTG ΛCC ACT ΛCG CAA ΛAT GTΛ CAΛ GGT TCA TAT CΛG Gly Ala Tyr Ala Pro Val Leu Cln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn 1,278 CGG GCA TAT CCT CCA CTA CTT CΛA CΛT TTT ΛGG T A TTA ΛAT GAT TCA ACA AAT Arg Thr Lys Lye His Thr Ala His Phe Ser Lys Lys Cly Clu clu Clu Λsπ Leu 1,296 ACA ΛCΛ AΛC AΛA CΛC ΛCA CCT CAT TIC TCΛ AAA AAA GCC CΛG CΛΛ CΛΛ AAC TTC Clu Cly Lau Cly Asn Gin Thr Lys Cln lie Val Clu Lys Tyr Λla Cys Thr Thr 1,314 CΛA CCC TTC CCA AΛT CΛΛ ACC ΛΛC CAA ΛTT GTA CAG AAA TAT GCΛ TCC ACC ACA Ar g II, ser Pro Asn Thr Ser Cln Cln Asn Phe Val Thr Gin Ar_ Ser Lya Arg 1,332 A CC ATA TCT CCT AAT ACA AGC CAG CAG AAT TTT CTC ACC CAA CCT AGT AAG ACA TABLE 1, continued Λ la L « u Lys Gin Phβ ArR Leu Pro Leu Glu Clu Thr Clu Leu Glu Lys Λrg He 1,350 CCT TTC AAA CAA TTC ΛGA CTG CCΛ CTA CΛΛ CΛΛ ΛCA CΛΛ CTT GΛA ΛΛΛ ΛCG ΛTA lie" Val Λβp Asp Thr Ser Thr Cln Trp Ser Lys Λsn .KT Lys Hla I.cu Thr Pro- 1,368 ATT f!TG GAT GAC ΛCC TCA ΛCC CAC TCG TCC AΛΛ ΛΛC ATC ΛAA CAT TTG ΛCC CCG Sβr Thr Leu Thr Cln Ilo Asp Tyr Asn Clu Lys Glu Lys Gly Ala lie Thr Gin 1/386 ACC ACC CTC ACA CAG ATA CAC TΛC ΛAT CAG AΛG GAC ΛAA CGG CCC ΛTT ΛCT CAG Ser Pro Leu Ser Asp Cya Leu Thr Arg Sβr His Ser I ; Pro Gin Ala Aβn Arg 1,404 TCT CCC TTA TCA CAT TCC CTT ACG AGG ACT CAT ACC ATC CCT CAA CCA ΛAT ACA Sβr Pro Leu Pro lie Ala Lys Val Ser Ser Phu Pro Sβr llα Arg Pro lie Tyr 1/422 TCT CCA TTA CCC ATT GCA AAG GTA TCA TCA TTT CCA TCT ATT ΛGA CCT ATA TAT Leu Thr Arg Val Leu Phe Gin Asp Aβn Ser Ser Hla Leu Pro Ala Ala Ser Tyr 1 440 CTG ACC ACC GTC CTA TTC CAA CAC AAC TCT TCT CAT CTT CCA CCA CCA TCT TAT Arg Lys Lys Asp Sβr Cly Val Cln Glu Ser Ser Hlβ Phe Leu Cln Gly Λla Lys 1/458 ACA AAC AAA GAT TCT CGG CTC CAA CAA ACC AGT CΛT TTC TTA CΛA GCA CCC AΛ Cys Λsn Asn Leu Ser Leu Λla He Leu Thr Leu Glu MET Thr Cly Λsp Cln Arp, 1/476 AAA ΛAT AAC CTT TCT TTA GCC ΛΓΓ CTΛ ACC TTG CΛG ATC ACT CCT CAT CΛA ACA Clu Val Cly Sβr Leu Gly Thr Ser Ala Thr Λsπ Sor Val Thr Tyr Lys Lys Val 1/494 GAG CTT GGC TCC CTG GGC ACA AGT CCC ΛCA AAT TCA CTC ACA TAC ΛΛC ΛAΛ GTT Clu Λsn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr Ser Gly Lys Val Glu 1/512 CAG AAC ACT CTT CTC CCG AAΛ CCA CΛC TTG CCC AAA ΛCΛ TCT CCC ΛΛΛ GTT CAA Leu Leu Pro Lye Val His lie Tyr Cln Lys Λap Leu Phβ Pro Thr Glu Thr Ser 1/530 TTC CTT CCA AAA CTT CAC ATT TAT CAG AAC CΛC CTA TTC CCT ΛCC CΛΛ ACT ΛCC Aβn Gly Sβr Pro Gly His Leu Asp Leu Val Clu Gly Ser Leu Leu Cln Cly Thr 1/548 AAT- CCC TCT CCT GGC CAT CTG CAT CTC CTC CAA CCC ACC CTT CTT CAG CCA ACA Glu Gly Λl* lie Lye Trp Aan Clu Ala Asn Arg Pro Gly Lys Val Pro Phβ I.eu 1/566 GAG GCA GCC ATT AAC TCG AAT GΛA CCA AAC ACA CCT CCΛ AAA CTT CCC TTT CTC Arg Val Ala Thr Glu Sβr Sβr Ala Lye Thr Pro Sor Lys Leu Leu Asp Pro Lou 1/5B4 ACA CTΛ CCA ACA CAA ACC TCT CCA AAG ΛCT CCC TCC ΛAC CTA TTC GAT CCT CTT Ala Trp Aap Asn Hlβ Tyr Gly Thr Cln lie Pro Lys Glu Clu Trp Lye Ser Gin 1» 602 CCT TCG GAT AAC CAC TAT CCT ACT CΛG ATΛ CCΛ AAA CAΛ CΛC TCG ΛΛΛ TCC CAΛ Clu Lys Sβr Pro Clu Lys Thr Ala Phe Lya Lys Lys Asp Thr He Leu ! r Leu 1/520 CAG AAG TCA CCA CAA AAA ACA CCT TTT ΛΛC ΛΛΛ AAG CAT ACC ATT TTC TCC CTC Aβn Ala Cys Glu Ser Λsn His Ala lie Λla Ala 11. ,\*n Glu Cly Cln Λsn Lys 1/638 λΛC CCT TCT CAA ΛCC AAT CAT CCΛ ΛTΛ CCΛ CCΛ ΛTΛ ΛΛT CAC GCA CAA ΛΛT ΛΛG Pro Glu lie Glu Val Thr Trp Ala Lys Gin Cly Arg Thr Glu Arg Leu Cys Ser 1/656 CCC CΛΛ ΛTA CAA CTG ΛCC TCG CCA ΛΛC CAA CCT A ! ACT CΛA M.C, (.TG TCC TCT Cln Ann Pro Pro Vnl Leu Lys Arg His Cln Arg Glu He Thr Λrg Tlir llir Leu 1,674 CAA AAC CCΛ CCA GTC TTC AAA CCC GAT CAA CCC CAA ATA ACT CCT ACT ACT CTT TABLE 1. continued C ln Ser A sp Gin Clu Glu Ho Asp Tyr Λsp Λsp Thr He Ser VΛI Glu MET Lys 1,692 C A C TCA C A T CΛA CAG CAΛ ATT GAC TAT GΛT CAT ACC ATΛ TCA GTT CAA ATG AAG Lys Clu A sp The Asp He Tyr Λsp Glu Asp Glu Asn Cln Ser Pro Arg Ser Phe 1,710 A AG C A A CAT TTT CAC ATT TAT CAT CAC CAT CΛΛ AAT CΛC ACC CCC CGC AGC TTT Cln Lys Lys Thr Arg His Tyr Phβ He Ala Ala Val Clu Arg Leu Trp Aap Tyr 1,728 CAA AAG AAA ACA CCA CAC TAT TTT ATT CCT GCΛ GTC GAG AGG CTC TGG CAT TAT Cly MET Ser Sβr Sβr Pro Hla Val Leu Arg Asn Arg Ala Gin Ser Cly Sβr Val 1,746 CCC ATC AGT ACC TCC CCA CAT CTT CTΛ ACA AAC ΛCC CCT CAG AGT CCC ACT CTC Pro Cln Phβ Lye Lys Val Val Phβ Cln Glu Phe Thr Asp Gly Ser Phβ Thr Gin 1,764 CCT CAG TTC AAG AAA GTT CTT TTC CAC CAA TTT ACT GAT CCC TCC TTT ACT CAG Pro Leu Tyr Arg Cly Clu Leu Λsn Glu His Leu Gly Leu Leu Gly Pro Tyr He 1,782 CCC TTA TAC CCT CCA CΛA CTA AAT CAA CΛT TTG GCA CTC CTC GCG CCA TΛT ΛTA Arg Ala Glu Val Clu Asp Asn He MET Val Thr Phe Arg Asn Gin Ala Sβr Arg 1,800 AGA CCΛ CAA CTT CAA CAT AAT ΛTC ATG GTA ACT TTC ACA AAT CAG CCC TCT CCT Pro Tyr Ser Phβ Tyr Ser Ser Leu He Sur Tyr Clu Glu Asp Cln Arg Gin Gly 1,818 CCC TAT TCC TTC TΛT TCT ACC CTT ATT TCT TAT CAG CAA CAT CAC ACG CΛA CGA Ala Clu Pro Arg Lys Asn Phβ Val Lys Pro Asn Glu Thr Lye Thr Tyr Phe Trp 1,836 GCA CAA CCT ACA AAA ΛAC TTT CTC AAG CCT AAT CAΛ ΛCC ΛAA ACT TΛC TTT TCC Lya Val Cln Hlβ His MET Ala Pro Thr Lys Aβp Glu Phβ Asp cys Lys Ala Trp 1,854 AAA GTC CAA CAT CAT ΛTC GCA CCC ACT ΛAA CΛT GAG TTT GAC TGC AAΛ CCC TCC Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Acp Val His Ser Gly Leu He Cly 1,872 GCT TAT TTC TCT CAT CTT GAC CTG CAA ΛΛΛ CAT GTC CAC TCA CCC CTG ΛTT CGA Pro Leu Leu V_l Cys His Thr Aan Thr Leu Asn Pro Ala Hie Gly Arg Cln Val 1/890 CCC CTT CTC CTC TCC CAC ACT AAC ACA CTG ΛΛC CCT CCT CAT CGG AGA CΛA CTG Thr Val Clu Clu Phβ Ala Leu Phβ Phe Thr He Phe Λsp Glu Thr Lys Ser Trp 1,908 ACA CTA CAG CAA TTT GCT CTG TTT TTC ACC ATC TTT CAT CΛC ΛCC AAA AGC TCG Thy Phe Thr Clu Aβn MET Clu Arg Λsn Cys Arg Ala Pro Cys Asn He Gin MET 1/926 TAC TTC ACT CAA ΛAT ATC CAA ACA AAC TCC ACG GCT CCC TCC AAT ATC CΛC ATG Clu Asp Pro Thr Phβ Lys Glu Asn Thr Arg Phe Hlβ Ala He Λβπ Cly Tyr He 1,944 CAA CAT CCC ACT TTT AAA CAG AAT TAT CGC TTC CAT GCA ATC ΛAT GCC TAC ATA MET Λ β p Thr Leu Pro Cly Leu Val MET Ala Gin Asp Gin Arg He Arg Trp Tyr 1,962 ATC GAT ACA CTA CCT CCC TTΛ CTA ΛIG CCT CAG GΛT CAΛ AGG ATT CGA TCG TAT Leu Leu Ser MET Cly Ser Λsn Clu Asn !!/.• fits Ser He Hlβ Phe Ser Gly Hts 1,980 CTC CTC ACC ΛTG CGC ACC AAT CAA AAC ATC CAT TCT ΛTT CAT TTC ACT CCΛ CAT Val Phe Thr Val Arg Ly.-. Lye Glu Clu Tyr Lys HET Ala l.eu Tyr Asn I.eu Tyr 1,998 CT C TTC ΛCT GTA CCA AAA AAΛ CAG GAG 1AT AΛΛ ΛTG CCΛ CTC TAC AAT CTC TAT Pr o Cly V-l Phe Clu Thr Vβl Glu MET Leu Pro Ser Lys Λla Cly He Trp Arg 2,016 CCA CCT CTT TTT CAC ACA GTC CAA ATC TTΛ CCA XCC AAA GCT CCA ATT TCC CGG TABLE 1, continued Va l Clu Cys Leu He Cly Clu H Leu His Λla Gly MITT Ser Thr Leu Phe Leu 2 / 034 CTG CAΛ TGC CTT ATT CCC CAG CAT CTA CAT GCT GCG ΛTG ΛCC ACA f TT TTT CTG Val Tyr Sβr Aβn Lys Cys Cln Thr Pro Leu Cly MET Ala Ser Gly His H e Λrg 2 ° 52 CTG TAC ACC AAT AAG TGT CAG ACT CCC CTG CCA ATG GCT TCT CCΛ CAC ATT AGA Λβp Phβ Cln He Thr Ala Ser Gly Cln Tyr Gly Cln Trp Ala Pro Lys Leu Ala 2 » 070 CAT TTT CAC ATT ACA GCT TCΛ CCA CAA TAT CCA CAG TGG CCC CCA ΛAC CTC CCC Arg Leu Hie Tyr Sβr Cly Ser He Asn Ala Trp Sβr Thr Lys Glu Pro Phβ Sβr 2 / ° 88 ACA CTT CAT TAT TCC GCA TCA ATC AAT GCC TCG AGC ACC AAG CAG CCC TTT TCT Trp He Lya Val Aβp Leu Leu Ala Pro MET He He Hlβ Cly He Lye Thr Gin 2 ° 6 TCC ATC AAG CTG CAT CTG TTG GCA CCA ATC ATT ATT CAC GCC ATC AAG ACC CAG Cly Ala Arg Cln Lye Phβ Sβr Ser Leu Tyr He Ser Gin Phβ He He MET Tyr 2 ' 124 CGT GCC CCT CAG AAG TTC TCC AGC CTC TAC ATC TCT CAC TTT ATC ATC ATG TAT 2 142 Sβr Leu Aβp Cly Lye Lye Trp Gin Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu ' AGT CTT CAT GCG AAG AAG TGG CAG ACT TAT CCA CCA AAT TCC ACT GGA ACC TTΛ MET Val Phβ Ph« Gly Asn Val Asp Sβr Ser Gly He Lys His Asn He Phe Λsn 2 / 160 ΛTG CTC TTC TTT CGC AAT CTC CAT TCA TCT CCC ATA AAA CΛC AAT ATT TTT AAC 2 , 173 Pro Pro He He Ala Arg Tyr He Arg Leu Hla Pro Thr His Tyr Ser He Arg CCT CCA ATT ATT CCT CGA TAC ATC CGT TTG CΛC CCA ACT CAT TAT ΛCC ΛTT CCC 2 , 196 Ser Thr Leu Arg MET Clu Leu MET Gly Cys Asp LPU ΛSU Ser Cys Ser MET Pro ACC ACT CTT CGC ATG GAG TTC ATG CCC TGT CAT TTA AAT AGT TCC ΛCC ATG CCA 2 , 214 Leu Cly MET Clu Ser Lys Ala H e Ser Asp Ala Cln He Thr Ala Ser Ser Tyr TTG GCA ATG CAG ACT AAA GCA ATA TCA GAT GCA CAC ATT ACT CCT TCA TCC TAC Phβ Thr Aβn MET Phβ Ala Thr Trp Sβr Pro Ser Lys Ala Arg Leu His Leu Cln 2 ' 232 ITT ACC AAT ATG TTT GCC ACC TCC TCT CCT TCA AM GCT CCΛ CTT CAC CTC CAA Gly Arg Sβr Asn Ala Trp Arg Pro Cln Val Λsn Λsn Pro Lys Glu Trp Leu Gin 2 ' 250 CCC ACG AGT ΛAT CCC TCC ACA CCT CAG CTC ΛAT AAT CCA AAΛ CAG TCG CTG CAA Val Asp Phβ Cln Lye Thr MKT Lye Val Thr Cly Val Thr Thr Gin Cl y Val Lys 2 ' 268 GTC CAC TTC CAG AAG ACA ΛTC ΛAA GTC ΛCA CGΛ CTA ACT ACT CAG CCA GTA ΛΛA Sβr Leu Leu Thr Sβr MET Tyr -Val Lys Glu Phβ Leu 11- Sor Sβr Ser Cln Λsp 2 / 286 TCT CTG CTT ACC ACC ATC TAT CTC AΛC GAG TTC CTC ATC TCC ΛCC ACT CΛΛ CAT Gly Hl β Cln Trp Thr Lau Phβ Phβ Cln Asn Cly Lys V.il Lys VΛ I Phe G in G ly 3 0 CCC CAT CAG TCG ACT CTC TTT TTT CΛG AAT CCC AAA CTA ΛAC CTT TTT CAG CCΛ Λsn Cln Asp Ser Phβ Thr Pro Va l Val Aβn Sor Leu Λsp Pro Tro Leu Leu Thr 2 322 AAT CΛA CAC TCC TTC ACA CCT CTC CTG AAC TCT CTA CΛC CCA CCG TTΛ CTG ΛCT Arg Tyr Leu Arg He His Pro Gin Sβr Trp Val Hi s Gi n T ie Λ l . Lou Λrι» MKT 2 / 340 CCC TAC CTT CCΛ ATT CΛC CCC CAG ACT TCG CTC CΛC CΛG ATT CCC CTG ΛCC ΛTC Glu Val Lau Cly Cys Glu Ala Gin Λsp Leu Tyr End 2 , 352 CAC CTT CTC GCC TGC CAG CCA CAC CAC CTC TAC TCA GGGTCCCCΛCTCCATCCCACCTCCC AGTC CCCTCACCTCTCCCTCCTCACCTCCACCCCATCTC_:CCCTCCCTGGCTTCCTTCTACCTTrCTCCTAAATCCTACCAGACACTCCCTTC AACCCTCCTGAATTAACTATCATCAGTCCTCCAr 1 Σ "1 ICGTCCCGCCCCAGCAGCCTCCATCCATTTTAACTrAACTCTTACCTATT TTCrCCΛCCTCCTCCCACΛ 1000 followed by the amino acid sequence of Asp-1582 to Arg— 1708. That compound thus comprises the polypeptide sequence of Ala-20 to Pro-1000 covalently linked by a peptide bond to amino acids Asp-1582 to Tyr-2351. Another exemplary compound contains a region X comprising the amino acid sequence Ser-760 to Thr-778 followed by the sequence Pro-1659 to Arg-1708. That compound thus comprises the polypeptide sequence Ala-20 to Thr-778 covalently linked by a peptide bond to the sequence Pro-1659 through Tyr-2351. Still another exemplary compound contains a region X comprising the amino acid sequence Ser-760 to Thr-778 followed by the sequence Glu-1694 to Arg-1708. That compound thus comprises the polypeptide sequence Ala-20 to Thr-778 covalently linked by a peptide bond to amino acids Glu-1694 through Tyr-2351. These exemplary compounds are depicted schematically in Table 2. The amino acid sequence represented by X should be selected so that it does not substantially reduce the procoagulant activity of the molecule, which activity can be conveniently assayed by conventional methods. Compound (2) of Table 2 is a presently preferred embodiment. The procoagulant protein may be produced by appropriate host cells transformed by factor VIII:C DNA which has been specific¬ ally altered by use of any of a variety of site-specific muta- genesis techniques which will be familiar to those of ordinary skill in the art of recombinant DNA. The starting materials may be a DNA sequence which codes for the complete factor VIII:C molecule, e.g., the complete human factor VIII:C as shown in Table 1, a truncated version of that sequence, or it may comprise segments of that DNA sequence, so long as the starting materials contain at least sufficient DNA to code for the amino acid sequences of the desired polypeptide. TABLE 2: EXEMPLARY COMPOUNDS A-X-B Compound Amino Acid Sequence X Del (human (Ala n —^ yr ^ -,... factor 20 2351) (Ser 760~ Arg l708 ) VIII:c) 1 (Ala 20 " - >PrO 1000 ) - ) 2 (Ala 20 → hr 778 ) -(Pro 1659 →Tyr 2351) (Ser^→Thr.^) - ( Pro 1659 ->Arg 17()8 ) (Ala 20 -^T r 778 ) -(Glu 1694 -> yr 2 3 51 ) (Ser 760 "*>Thr 778 ) -(Glu 1694 →Arg 1708 ) A and B are as defined, supra; "-" represents a peptide bond; "—^»" indicates a polypeptide sequence inclusive of the specified amino acids; amino acid numbering corresponds to the numbering of the sequence depicted in Table 1; and "deletion" indicates the number of amino acids deleted relative to human factor VIII:c. The procoagulent proteins of the present invention, in addition to lacking a substantial amino acid segment of human factor VIII:C, also have fewer potential N-glycosylation sites than human factor VIII. Preferably, at least one N-glycosylation site has been deleted. More preferably, 18 of the 25 potential N-glycosylation sites are not in the molecule. In still more preferred embodiments, up to 19 of the 25 potential N-glycos¬ ylation sites are removed. While not wishing to be bound by theory, it is presently believed that the antibodies to factor VIII:C which are directed to antigenic determinants contained in the protein segment deleted in accordance with this inven¬ tion, i.e., in the amino acid segement itself or in the carbo¬ hydrate portion of the glycosylated protein, will not neutralize the procoagulant proteins of the present invention. Moreover, the fact that the procoagulants of the present invention lack many of the sites for non-human glyσosylation by the non-human mammalian or other cells used.to produce the proteins is also belived to reduce the antigeniσity of that protein, and lessen the likelihood of developing antibodies to the procoagulants. This may enable facilitating the treatment of patients in need of procoagulant therapy. I contemplate that my compounds can be produced by recombinant DNA techniques at a much lower cost than is possible for pro- duction of human factor VIII. The host organisms should more efficiently process and express the substantially simpler molecules of this invention. The compounds of this invention can be formulated into pharmaceu- tiσally acceptable preparations with parenterally acceptable vehicles and excipients in accordance with procedures known in the art. The pharmaceutical preparations of this invention, suitable for parenteral administration, may conveniently comprise a sterile lyophilized preparation of the protein which may be reconsti- tuted by addition of sterile solution to produce solutions preferably isotonic with the blood of the recipient. The prepar¬ ation may be presented in unit or multi-dose containers, e.g. in sealed ampoules or vials. Their use would be analogous to that of human factor VIII, appropriately adjusted for potency. One method by which these proteins can be expressed is by use of DNA which is prepared by cutting a full-length factor VIII:C DNA with the appropriate restriction enzymes to remove a portion of the DNA sequence that codes for amino acids 760 to 1708 of human factor VIII:C. The cut DNA is then ligated with an oligonuσleotide that resects the cut DNA and maintains the correct translational reading frame. Preparation of the cDNA has been set forth in detail in U.S. Patent Applications Serial Nos. 546,650 and 644,086, supra. A pSP64 reσombinant clone containing the nucleotide • sequence depicted in Table 1, designated as pSP64-VIII, is on deposit at the American Type Culture Collection under Accession Number ATCC 39812. Restriction endonucleases are used to obtain cleavage of the human factor VIII:C cDNA, hereinafter the DNA source sequence, at appropriate sites in the nucleotide sequence. Unless otherwise noted, restriction endonucleases are utilized unde the conditions and in the manner recommended by their commercial suppliers. The restriction endonucleases selected herein are those which will enable one to excise with substantial speci¬ ficity sequences that code for the portion of the facto VIII:C molecule desired to be excised. BamHI and Sad are particularly, useful endonucleases. However, the skilled artisan will be able to utilize other restriction endonucleases chosen by conventional selection methods. The number of nuσleotides deleted may vary but care should be taken to insure that the reading frame of the ultimate cDNA sequence will not be affected. The resulting DNA fragments are then purified using conventional techniques such as those set forth in Maniatis et al., Molecular Cloning. A Laboratory Manual (Cold SpringHarbor Laboratory 1982) the disclosure of which is incorporated herein by reference, and Proσ. Natl. Acad. Sci. 76:615-619 (1979). The purified DNA is then ligated to form the sequence encoding the polypeptide of the preferred invention. When necessary or desirable, the ligation may be within an oligonucleotide that resects the cut DNA and maintains the correct translational reading frame using standard ligation conditions. Ligation reactions are carried on as described by Maniatis et al., supra at 2453-6 using the buffer described at page 246 thereof and using a DNA concentration of 1-100 ug/ml, at a temperature of 23"C for blunt-ended DNA and 16°C for "sticky ended" DNA. The following double-stranded oligonucleotide is useful when there is BamHI/- Saσl deletion such as described infra, 5" P-CATGGACCG-3• 3-TCGAGTACCTGGCCTAG 5• ; but other oligonucleotides can be selected by the skilled artisan depending upon the deletions made and reaction conditions. The DNA sequences encoding the novel procoagulant polypeptides can, in addition to other methods, be derived from the sequence of human factor VIII:C DNAby application of oligonucleotide-med- iated deletion mutagenesis, often referred to as "loopout" muta- genesis, as described for example inMorinaga, Y. et al. Biotech¬ nology.. 2 : 636-639 (1984) . The new DNA sequences containing the various deletions can then be introduced into appropriate vectors for expression in mammalian cells. The procoagulant activity produced by the transiently transfected or stably transformed host cells may be measured by using standard assays for blood plasma samples. The eukaryotic cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art. The components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See ^ Kaufman et al., J. Mol. Biol.. 159; 51-521 (1982) ; Kaufman, Proc. Natl. Acad. Sci. 82: 689-693 (1985) . Established cell lines, including transformed cell lines, are suitable as hosts. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as haematopoeitic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is do inantly acting. The host cells preferably will be established mammalian cell lines. For stable integration of the vector DNA into chromosomal DNA, and for subsequent amplification of the integrated vector DNA, CHO (Chinese hamster ovary) cells are presently preferred. See U.S. Patent 4,399,216. Alternatively, the vector DNA could include all or parts of the bovine papilloma virus genome (Lusky et al., Cell. 36: 391-401 (9184) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Other usable mammalian cell lines include HeLa, COS-1 monkey cells, melanoma cell lines such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cells lines and the like. Stable transformants then are screened for expression of the procoagulant product by standard immunological or enzymatic assays. The presence of the DNA encoding the procoagulant proteins may be detected by standard procedures such as Southern blotting. Transient expression of the procoagulant genes during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by enzymatic or immunologic assay of the proteins in the culture medium. The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary, and should not be taken as limiting the true scope of the present invention, as described in the claims. EXAMPLE 1 10 ug. of the plasmid pACE, a pSP64 (Promega Biotec, Madison, Wis. ) derivative, containing nucleotides 562-7269 of human factor VIII:C cDNA (nucleotide 1 is the A of the ATG initiator meth- ionine codon) was subjected to partial BamHI digestion in lOOul containing 50mM Tris.HCl ph 8.0, 50mM MgCl 2 , and 2.4 units BamHI (New England Biolabs) for 30 minutes at 37 β C. The reaction was terminated by the addition of EDTA to 20mM and then extracted once with phenol, once with chloroform , ethanol precipitated and pelleted by centrifugation. DNA was redis- solved, cleaved to completion in 50ul using 40 units Sad for 1.5 hours at 37 β C. DNA was then electrophoresed through a buffered 0.6% agarose gel. An 8.1 kb fragment corresponding to the partial BamHI-SacI fragment of pACE lacking only the sequence corresponding to nucleotides 2992-4774 of the factor VIII:C sequence was purified from the gel using the glass powder technique described in Proc. Nat. Acad. Sci. 76; 615-619 (1979) . Purified DNA was ligated with 100 pmoles of the following double-stranded oligonucleotide 5»P-CATGGACCG-3 • 3 '-TCGAGTACCTGGCCTAG 5• using standard ligation conditions. The DNA sequence removed " represents the deletion of 584 amino acid sequence beginning with amino acid 998 and continuing through 1581. The oligo¬ nucleotide inserted, however, encodes amino acids corresponding to 998-1000. Therefore, the polypeptide encoded contains deletion of 581 amino acids. DNA was then used to transform competent |__ coli bacteria, and DNA from several a picillin resistant transformants was analyzed by restriction mapping to identify a plasmid harboring the desired SacI-BamHI deletion mutant. DNA from this plasmid was digested to completion with KpnI., which cleaves the plasmid uniquely at nucleotide 1816 of the factor VIII:C coding se- quence. This DNA was ligated with a Kpnl DNA fragment containing nucleotides 1-1815 of factor VIII:C DNA and a synthetic Sail site at nucleotides -11 to -5 and then used to transform competent E. coli bacteria. Plasmid DNA was isolated and oriented by restriction mapping to identify a plasmid, pBSdK, containing the correct 5' to 3' orien¬ tation of the Kpnl insert. Sail digestion, which excises the entire polypeptide coding region from the plasmid, was performed and the DNA electrophoresed through a buffered 0.6% agarose gel. The 5.3Kb Sail fragment was purified from the gel as described above. This DNA fragment was ligated with Xhol cut pXMT2 DNA to give rise to plasmid pDGR-2. pXMT2 is a plasmid capable of expressing heterologous genes when introduced into mammalian cells such as the COS-1 African Green Monkey kidney cell line, and is a derivative of the expression vectors described in Kaufman, supra at 689-93. The expression elements are the same as described for plasmid pQ2 except that it contains a deletion of the adenovirus major late promoter extending from -45 to +156 with respect to the.transcription start site of the adenovirus major late promoter. mRNA expression in pXMT is driven by the SV40 late promoter. The bacterial replicon, however, has been substituted to render bacteria containing the vector resistant to ampiσillin rather than tetracyσline. pXMT2 contains a unique Xho I site at a position which allows for expression of inserted cDNA from the SV40 late promoter. This Xho I site is convenient for inserting factor VIII:C cDNA constructs since these are flanked by Sail sites. Restriction mapping of transformants identified a plasmid, pDGR-2, containing the correct 5• to 3• orientation of the polypeptide coding sequence relative to the direction of transcription from the SV40 late promoter. pDGR-2 is on deposit at the American Type Culture Collection under Accession number 53100. EXAMPLE 2 Other novel procoagulant proteins maybe obtained from constructs produced by oligonucleotide mediated deletion mutagenesis, using for example the "loopout" mutagenesis techniques as described in Morinaga et al. , supra. The deletion mutagenesis is performed using expression plasmid pDGR-2 or any other appropriate plasmid or bacteriophage vector. Other methods for oligonucleotide mediated mutagenesis employing single stranded DNA produced with M13 vectors and the like are also suitable. See Zoller et al., Nucl. Acids Res. 10: 6487-6500 (1982) . For example, these deletions can be produced using the oligonucleotides (A) 5• AAAAGCAATTTAATGCCACCCCACCAGTCTTGAAACGCCA (B) 5' AAAAGCAATTTAATGCCACCGAAGATTTTGACATTTATGA to cause deletions in factor VIII:C cDNA from nucleotides (A) " 2334 to 4974 or (B) 2334 to 5079. The proteins encoded by these constructs contain deletions of (A) 880 and (B) 915 amino acids relative to Factor VIII:C. The deleted constructs are tested directly, or after subcloning into appropriate expression vectors, in order to determine if the novel proteins possess procoagulant activity. Procoagulant activity was assayed as described in Examples 3 and 4. EXAMPLE 3 Expression of Procoagulant Molecules in COS Monkey Cells The expression plasmids containing the modified cDNA's prepared as in Examples 1 or 2 and the full-length cDNA, pXMT-VIII, were introduced into COS-1 cells via the DEAE-dextran trans- feetion protocol. So payrac and Dana 1981, Proc. Natl. Acad. Sci. 78: 7575-7578. Conditioned media was harvested 48 hours post-transfeetion and assayed for factor Vlll-type activity as described in Toole et. al., 1984, Nature 312:342-347. The results of the experiment are summarized in Table 3. Both plasmids containing the modified cDNAs yielded procoagulant activity and, moreover, the activity was greater than that obtained using wild type cDNA. From these data it was concluded that removal of up to 880 amino acids (95,000 daltons) in a defined domain of human factor VIII does not destroy cofactor activity. Furthermore, these abridged procoagulant proteins retain their ability to be activated by thrombin. TABLE 3: EXPRESSION OF ABRIDGED FACTOR VIII MOLECULES # amino chromogenic Clotek acids activity activity plasmid deleted fmUml -1 ) -Ila +IIa (fold) No DNA 0 pXMT-VIII 15:1 450 pDGR-2 581 114 250 5750 (23X) pLA-2 880 162 330 9240 (28X) The plasmids indicated were transfected into COS cells and 48 hr. post-transfection the conditioned media taken for assay by the Kabi Coatest factor VIII:C method (chromogenic activity) and by the one-stage activated partial thro boplastin time (APTT) coagulation assay (Clotek activity) using factor VIII:C deficient plasma as described (Toole, Nature 1984) . For thrombin (Ila) activation, samples were pretreated 1-10 min, with 0.2 units/ml thrombin (Ila) at roomtemperature. Activation coefficients are provided in parentheses. Activity from media from the wild-type (pXMT-VIII) transfection was too low to directly measure Clotek activity before thrombin activ¬ ation. From other experiments where the wild type factor VIII activity was concentrated, it was demonstrated to be approximately 30-fold aσtivatable. EXAMPLE 4 Expression of Procoagulant Molecules in CHO Cells A) Expression of pDGR-2 The procoagulant expression vector containing a deletion (relative to the Factor VIII:C cDNA) of 581 amino acids (pDGR-2) was transfected with plasmid pAdD26SV(A) #3 (10 ug pDGR-2:l ug pAdD26SV(A) #3) by CaP0 4 coprecipitation into CHO DHFR deficient 0 cells (DUKX-Bll) and transformants isolated and grown in increasing concentrations of MTX as described by Kaufman et. al. , (1985) . One transfor ant designated Jl exhibited the following activities as a function of resistance to increasing concen¬ trations of MTX. 15 uM MTX mUnits/ml/dav/10 6 cells* 0 1.46 0. 02 322 0. 1 499 20 B) Expression of pLA-2 The procoagulant expression vector containing a deletion of 880 amino acids (pLA-2) was introduced into CHO DHFR deficient 25 cells (DUKX-Bll, Chasin and Urlaub, PNAS 77: 4216-4220, 1980 by protoplast fusion as described (Sandri-Goldin et. al., Mol. Cell. Biol. 1 : 743-752) . After fusion, fresh medium containing 100 ug/ml of kanamyσin, and 10 ug/ l of each of thymidine, adenosine, deoxyadenosine, penicillin, and streptomycin and 3 0 10% dialyzed fetal calf serum was added to each plate. The kanamycin was included to prevent the growth of any bacteria which had escaped conversion to protoplasts. Four days later the cells were subcultured 1:15 into alpha-media with 10% dialyzed fetal calf serum, penicillin, and streptomycin, but 35 lacking the nucleosides. Colonies appeared after 10-12 days after subculturing cells into selective media. A group of B transformants were pooled and grown in sequentially increasing concentrations of MTX starting at 0.02 uM with steps to 0.1, 0.2, and 1.0 uM MTX. Results of factor Vlll-type activity in cells resistant to increasing concentrations of MTX is shown below. UM MTX mUnits/ml/dav/10 6 cells* 0 16 0 . 02 530 0.2 . 1170 1. 0 1890 * Factor VIII activity was determined by the Kabi Coatest factor VIII:C method (chromogenic activity).

Claims: What is claimed is: 1. A protein exhibiting procoagulant activity having the amino acid sequence: A-X-B wherein region A represents the polypeptide sequence Ala-20 through Arg-759 substantially as shown in Table 1; region B represents the polypeptide sequence Ser-1709 through Tyr 2351 substantially as shown in Table 1; and region X represents a polypeptide sequence comprising up to 949 amino acids substan¬ tially duplicative of sequences of amino acids within the sequence Ser-760 through Arg-1708 of Table 1, wherein the amino terminus of X is covalently bonded through a peptide bond to the the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B. 2. A protein of claim 1 comprising the amino acid sequence Ala-20 through Pro-1000 followed by Asp-1582 through Tyr-2351 substantially as shown in Table 1 wherein Pro-1000 is covalently bonded by a peptide bond to Asp-1582. 3. A protein of claim 1 comprising the amino acid sequence Ala-20 through Thr-778 followed by Pro-1659 through Tyr-2351, substantially as shown in Table 1, wherein Thr-778 is covalently bonded by a peptide bond to Pro-1659. 4. A protein of claim 1 comprising the amino acid sequence Ala-20 through Thr-778 followed by Glu-1694 through Tyr-2351, substantially as shown in Table 1, wherein Thr-778 is covalently bonded by a peptide bond to Glu-1694. 5. A DNA molecule encoding the protein of claim 1. 6. A DNA molecule encoding the protein of claim 2. 7. A DNA molecule encoding the protein of claim 3. 8. A DNA molecule encoding the protein of claim 4. 9. A genetically engineered host cell containing, and capable of expressing, a DNA molecule encoding the protein of claim 1. 10. A genetically engineered host cell of claim 9 wherein the host cell is a mammalian, yeast or bacterial cell. 11. A method for producing a protein exhibiting procoagulant properties which comprises culturing a genetically engineered cell of claim 9 under suitable conditions permitting expression of the protein. 12. Apharmaceuticalpreparationuseful fortherapeutictreatment of Hemophilia A comprising a sterile preparation of a protein of claim 1 in admixture with a pharmaceutically accepted carrier. 13. Apharmaceutical preparationuseful fortherapeutictreatment of Hemophilia A comprising a sterile preparation of a protein of claim 2 in admixture with a pharmaceutically accepted carrier. 14. A pharmaceutical preparation useful for therapeutic treatment of Hemophilia A comprising a sterile preparation of a protein of claim 3 in admixture with a pharmaceutically accepted carrier. 15. A pharmaceutical preparation useful for therapeutic treatment of Hemophilia A comprising a sterile preparation of a protein of claim 4 in admixture with a pharmaceutically accepted carrier. 16. A method of treating Hemophilia A comprising administering to a patient an effective dose of the preparation of claim 12. 17. A method of treating Hemophilia A comprising administering to a patient an effective dose of the preparation of claim 13. 18. A method of treating Hemophilia A comprising administer¬ ing to a patient an effective dose of the preparation of claim 14. 19. A method of treating Hemophilia A comprising administering to a patient an effective dose of the preparation of claim 15.

Inventors: TOOLE JOHN J JR, TOOLE, JOHN, J., JR.
Applicants: GENETICS INST, GENETICS INSTITUTE, INC.
Application year: 1986
Main classification: C12P21
IPC codes: A61K38, C07K14