Bovine Factor VII Research Articles

ChemInform Abstract: Synthesis of New Serine-Linked Oligosaccharides in Blood-Clotting Factors VII and IX and Protein Z. The Syntheses of O-α-D- Xylopyranosyl-(1 → 3)-D-glucopyranose, O-α-D- Xylopyranosyl-(1 → 3)-O-α-D-xylopyranosyl-(1 → 3)- D-glucopyrano

The di- and trisaccharide sequences and their conjugates with serine recently identified in the first epidermal growth factor-like domain of bovine and human blood-clotting factors VII, IX, and protein Z were synthesized in order to elucidate their biological function. The disaccharide α-D-Xylp(1→3)-D-Glcp was prepared from 2,3,4-tri-O-benzyl-α-D-xylopyranosyl fluoride and allyl 2,4,6-tri-O-acetyl-α-D-grucopyranoside (9a) using tin(II) chloride and silver perchlorate as glycosylating reagents. The trisaccharide α-D-Xylp(1→3)-α-D-Xylp(1→3)-D-Glcp was prepared from O-(2,3,4-tri-O-benzyl-α-D-xylopyranosyl)-(1→3)-2,4-di-O-benzyl-α,β-D-xylopyranosyl fluoride and 9a with the same reagents. Their conjugates with serine, α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser and α-D-Xylp(1→3)-α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser, were synthesized by the glycosylation of N-benzyloxycarbonyl-L-serine benzyl ester with the corresponding glycosyl trichloroacetimidates of the above saccharides, followed by removal of the protecting groups. T...

Identification of regions of bovine factor VII essential for binding to tissue factor.

Initiation of the extrinsic blood coagulation pathway is mediated by a complex formed between plasma-derived factor VII/VIIa and cell-derived tissue factor (TF). To identify the site(s) of interaction, zymogen VII and VIIa were enzymatically and chemically modified, and their affinities for TF were estimated by measuring their inhibitory effects on the amidolytic activity enhanced after formation of the VIIa-TF complex. We found that the VIIa-light chain (Ki = 3.5 x 10(-7) M) and its fragment consisting of the gamma-carboxyglutamic acid (Gla)-domain and the first epidermal growth factor (EGF)-like domain (Gla-EGF1 peptide; Ki = 1.0 x 10(-6) M) have an affinity for TF. Therefore, one of the binding sites of VII with TF is probably located in the Gla-EGF1 region. On the other hand, a dansyl-Glu-Gly-Arg chloromethyl ketone-treated Gla-domainless VIIa (Ki = 0.7 x 10(-7) M) showed a high affinity for TF, whereas the corresponding Gla-domainless VII similarly treated showed no binding potential, thereby indicating that binding site(s) other than in the Gla-EGF1 region are present in VIIa but not in VII. Acetylation or carbamylation of the alpha-amino group of the NH2-terminal Ile-153 of VIIa resulted in the loss of binding affinity for TF; such modifications convert VIIa into a zymogen-like inactive form by destroying the salt bridge between Ile-153 and Asp-343 in VIIa. The rate of carbamylation of VIIa was reduced in the presence of TF. Protection of the alpha-amino group of Ile-153 from carbamylation after complex formation was consistent with salt bridge formation between Ile-153 and Asp-343 in the VIIa-TF complex. Therefore, binding of TF with the heavy chain of VIIa may induce a conformational change that brings the alpha-amino group of Ile-153 close to the beta-carboxyl group of Asp-343 to make a stable salt bridge.

Complete nucleotide sequence of the cdna encoding rabbit coagulation factor VII

Rabbit coagulation factor VII cDNA has been cloned and sequenced. Two rabbit liver cDNA libraries in lambda gtll were screened using both random-primed, 32P-labelled, factor VII cDNAs and polymerase chain reaction technology. Three overlapping cDNA clones were isolated comprising a sequence of 1572 base pairs of DNA encoding an open-reading-frame of 443 amino acids. Mature circulating rabbit factor VII is predicted to contain 404 amino acids, two less than its human counterpart and three less than bovine factor VII.

Monoclonal Antibody (VII-M31) to Bovine Factor VII: A Specific Epitope in the γ-Carboxyglutamic Acid Domain1

A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.

The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor.

Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This finding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, we synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues 195-206, 263-274, and 314-326. Peptide 195-206 inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIIa and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 microM. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor VIIa interaction with tissue factor or factor VIIa-mediated activation of factor X. Our data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.

Identification of a Disaccharide (Xyl-Glc) and a Trisaccharide (Xyl2-Glc) O-Glycosidically Linked to a Serine Residue in the First Epidermal Growth Factor-like Domain of Human Factors VII and IX and Protein Z and Bovine Protein Z

We have recently described a unique trisaccharide linked to a serine residue in the first epidermal growth factor-like domains of bovine blood coagulation factors VII (Ser-52) and IX (Ser-53) (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The sugar chain identified in these clotting factors consists of 1 mol of hexose (glucose (Glc] and 2 mol of pentose (xylose (Xyl]. We report here that human factors VII and IX and protein Z and bovine protein Z also contain such carbohydrate moieties linked to a serine residue at the same position found in bovine factors VII and IX. A glycopeptide derived from each of these proteins was subjected to amino acid sequence and component sugar analyses and fast atom bombardment mass spectrometric analysis. The results indicate that the glycopeptide derived from human factor IX contains 1 mol each of Glc and Xyl. The reducing end of this disaccharide was identified as Glc by analyzing the disaccharide generated by hydrazinolysis. In contrast, human factor VII and protein Z yielded two different glycopeptides which contained Glc and Xyl at molar ratios of 1:1 and 1:2, respectively, suggesting microheterogeneity of these O-linked sugar chains. Bovine protein Z glycopeptide contained 1 mol of Glc and 2 mol of Xyl. These sugar compositions were confirmed by analyses of the intact proteins. In relation to the trisaccharide sugar chain previously discovered in bovine factors VII and IX, these findings indicate the existence of a Xyl2-Glc-Ser and a Xyl-Glc-Ser structure in the first epidermal growth factor-like domains of human factors VII and IX and protein Z in addition to that of bovine protein Z. Whether these carbohydrate moieties contribute to the biological activities of these proteins is unknown.

Bovine factor VII. Its purification and complete amino acid sequence.

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).

Affinity purification of human tissue factor: interaction of factor VII and tissue factor in detergent micelles.

Tissue factor, a known initiator of blood coagulation, was found to be active in Triton X-100. A system consisting of tissue factor, factor VIIa, calcium ions, and coagulation factor X generated activated factor X at an appreciable rate. Based on this observation, we coupled human and bovine factor VII to a solid support. Each column bound tissue factor, solubilized in Triton X-100, in a species-specific manner. These interactions required calcium ions; when the columns were washed with detergent containing calcium ions, no tissue factor was eluted. When calcium ions were omitted from the eluant, tissue factor emerged as a sharp peak. Human tissue factor was extracted from an acetone brain powder into 2% Triton X-100. This extract, made 10 mM in CaCl2, was passed over a factor VII column. Human factor VII (1.2 mg) was coupled to 30 ml of Affi-Gel 15. This column bound approximately equal to 15 micrograms of human tissue factor. The eluted material was approximately equal to 25% pure. Final purification was achieved by gel filtration after chymotryptic digestion of contaminants. The tissue factor activity was stable to this treatment. The molecular weight determined by sodium dodecyl sulfate/PAGE (approximately equal to 46,000) was also unchanged by chymotrypsin. The final material was a single band on PAGE, demonstrated similar resistance to tryptic and chymotryptic digestion as bovine tissue factor, and had approximately the same specific coagulant activity as the previously purified bovine material. Tissue factor was also purified from human placenta, yielding a similar protein. A partial 28-residue sequence of the latter has been obtained.

Immunoaffinity purification of bovine factor VII

Factor VII has been purified to homogeneity from bovine plasma by a procedure that includes affinity purification on an immunoadsorbent column. Recovery was determined by both coagulant assay and liquid scintillation counting, using 3H-factor VII as an internal standard. The purification factor calculated by both methods was approximately 120,000-fold, with a final yield of approximately 18%. Homogeneity was assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The material migrated as a single polypeptide chain of 53,000 daltons, and following activation by factor Xa, the one-chain zymogen was quantitatively converted to two-chain factor VIIa. Conversion of affinity-purified factor VII to factor VIIa resulted in up to a 119-fold activation of the coagulant activity, which is 2.7–4 times greater than the activatability reported for factor VII prepared by other methods. Zur et al. calculated that pure factor VII, uncontaminated by traces of factor VIIa, would be activated 123-fold upon conversion to factor VIIa. The close agreement between observed activatability of affinity-purified factor VII and the theoretical prediction suggests that we have isolated factor VII essentially free of factor VIIa. The purification data from three lots of bovine plasma yield an estimate for the plasma concentration of factor VII from 10.1 nM to 18.5 nM.

Purification and Properties of an Abnormal Blood Coagulation Factor IX (Factor IXBm)/Kinetics of Its Inhibition of Factor X Activation by Factor VII and Bovine Tissue Factor

An abnormal blood coagulation factor IX has been isolated from the blood of a hemophilia B patient with a variant of the disease (hemophilia Bm) characterized by a normal concentration of factor IX antigen, negligible factor IX coagulant activity, and a prolonged prothrombin time with bovine tissue factor. The isolated protein (factor IXBm) had the same apparent molecular weight as normal factor IX (55,000) and the same mobility on two dimensional immunoelectrophoresis as normal factor IX. Factor IXBm underwent limited proteolysis induced by activated factor XI, in the presence of Ca2+ ions, or induced by the reaction product of tissue factor, factor VII and Ca2+ ions. A timecourse study showed that activated factor XI cleaved factor IXBm and factor IX at similar rates. However, in contrast to normal factor IX, the limited protelysis of factor IXBm did not generate procoagulant activity. In kinetic experiments purified factor IXBm behaved like a competitive inhibitor (Ki of 0.017 muM) of the activation of factor X by bovine tissue factor and factor VII. Normal factor IX was also found to inhibit the reaction but required a four-fold higher concentration to activate the same inhibitory effects as factor IXBm.

Antiserum against factor IX shortens the bovine thromboplastin coagulation time of human plasma.

Abstract The one-stage prothrombin time was determined with bovine thromboplastin after the addition of a high-titred sheep antiserum against human factor IX. In normal plasma and plasma from patients with hemophilia B and a normal amount of plasma factor IX antigen, the addition of antiserum resulted in a shortening of the prothrombin time. Serum from a sheep which had not been immunized had no such effect. The antiserum had no effect on the prothrombin time of plasma from patients with hemophilia B and no detectable plasma factor IX antigen (hemophilia B-). Incubation with antiserum also resulted in a shortening of the clotting time of normal plasma in the Thrombotest and had no effect on the clotting time of hemophilia B-plasma. Addition of purified factor IX to normal plasma increased the prothrombin time determined with bovine thromboplastin. The antiserum had no effect on the prothrombin time determined with human or rabbit thromboplastin. It is concluded that normal factor IX antigen and factor IX antigen from patients with hemophilia B+ probably act as inhibitors in the reaction between bovine thromboplastin, factor VII and factor X.

Activation of bovine factor VII by hageman factor fragments

During the early events of coagulation of human blood by the intrinsic pathway, factor XII is activated to a form which can activate factor XI, and is proteolytically fragmented to smaller species (30,000 daltons and 70,000 daltons) which have lost most of the ability to activate factor XI but which can activate prekallikrein rapidly. The effect of these fragments on factor VII was studied. It was found that these Hageman factor fragments promoted rapid proteolysis of one-chain factor VII to a more active two-chain form. The amino-terminal sequences of the chains of activated factor VII were found to be Ala- Asx-Gly- and Ile-Val-Gly-, the same as were earlier observed after activation of factor VII by activated factor X. This finding indicates that initiation of coagulation by the intrinsic pathway also primes the extrinsic pathway.

Activation of bovine factor VII (proconvertin) by factor XIIa (activated Hageman factor).

Bovine factor VII (proconvertin) is a plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. It has a molecular weight of 45 500 and is composed of a single polypeptide chain with an amino-terminal alanine residue. Factor VII is readily converted to factor VIIa by factor XIIa (activated Hageman factor) employing an enzyme to substrate weight ratio of 1:50. Factor VIIa is composed of a light and a heavy chain held together by a disulfide bond(s). The heavy chain, which is formed from the carboxyl-terminal region of the precursor, contains an amino-terminal sequence of Ile-Val-Gly-Gly-. The heavy chain also contains the active-site sequence of -Phe-Cys-Ala-Gly-Tyr-Thr-Asp-Gly-Thr-Lys-Asp-Ala-Cys-Lys-Gly-Asp-Ser-Gly-Gly-Pro-His-. This sequence is homologous with the active-site region of a number of plasma serine proteases. These data indicate that factor VII is a typical precursor of a serine protease which is converted to an enzyme by factor XIIa by the cleavage of a single, internal peptide bond.

Factor VIII and human platelet aggregation. I. Evience that the platelet aggregating activity of bovine factor VIII is a property of its 'carrier protein' subunit.

Bovine factor VIII is a potent inducer of aggregation of human platelets. Upon gel filtration of five-thousand-fold purified material in 0.5 M CaCl2, bovine factor VIII is separated into high and low molecular weight components; the former contains both a 'carrier protein' and platelet aggregating activity, the latter the procoagulant activity (low molecular weight factor VII, LMW-FVIII). Upon removal of Ca2+ ions, LMW-F VIII recombines with the 'carrier protein'. LMW-F VIII modifies aggregation by 'carrier protein', but not aggregation by undissociated bovine factor VII, adenosine-5'-diphosphate or adrenaline. This finding indicates that the platelet aggregating activity in indeed a property of the 'carrier protein'.

Mechanism of activation of bovine factor VII. Products of cleavage by factor Xa.

Coagulation Factor VII from bovine plasma is a glycoprotein containing a single peptide chain. The NH2-terminal sequence of Ala-Asx-Gly-Phe-Leu- is homologous with the NH2 termini of prothrombin, Factor IX, and the light chain of Factor X. Factor Xa in the presence of calcium ions and phospholipid cleaves Factor VII at an Arg-Ile bond in the sequence Arg-Ile-Val-Gly-Gly-, producing a two-chain molecule with at least 85 times the coagulant activity of single-chain Factor VII and a new NH2-terminal sequence homologous with the corresponding chains of thrombin, Factor IXa and Factor Xa. A second slower cleavage at an Arg-Gly bond destroys Factor VII activity. Bovine Factor VII, unlike prothrombin, Factor IX, and Factor X, is rapidly inhibited by diisopropylphosphorofluoridate (iPr2PF). [3H]iPr2PF is readily incorporated into one-chain, two-chain, and three-chain forms of Factor VII up to ratios of approximately 0.9 moles of [3H]diisopropylphosphate per mole of protein. The radioactive peptides generated from each form of [32P]iPr2PF-inhibited Factor VII by tryptic and thermolytic digestion were found to migrate together on paper electrophoresis. This indicates that the iPr2PF is incorporated stoichiometrically into the same specific site in each form.

6] Bovine factor VII

Bovine factor VII is a glycoprotein containing a single peptide chain with a molecular weight of 53,000. Factor VII in conjunction with tissue factor and calcium ions initiates coagulation by the “extrinsic” pathway, and may be assayed by its ability to correct the clotting time of factor VII-deficient plasma upon addition of tissue factor and calcium ions. Plasma congenitally deficient in factor VII is rare, but normal bovine plasma may be selectively rendered deficient in factor VII by the method of Nemerson and Clyne. The latter method derives from the fact that unactivated bovine factor VII is rapidly and irreversibly inhibited by diisopropyl phosphorofluoridate (DFP), in contrast to factor X and prothrombin. Since human factor VII is not readily inhibited by DFP, human plasma cannot be used for the preparation of factor VII-deficient plasma. The article elaborates on purification procedure, chemical properties of Bovine Factor VII and activation process.

Physical, Chemical and Immunological Studies on Bovine Factor VIII

Highly purified bovine factor VII prepared according to our technique and having a specific activity of 500 U/mg protein has been studied. The chemical analysis of the preparation revealed it to be composed of amino acids, lipids (8-10%) and carbohydrates (7%). The lipid moiety can be removed by chromatography. Different mechanism of inactivation of bovine factor VII and the possible molecular changes induced by the inactivating agents were studied. EDTA and EGTA provoke a weakening of the bonds linking the structural elements of the molecule, which allows for the separation of two different components of the molecule by gel filtration. Upon treatment with thrombin, the carbohydrate content of bovine factor VIII decreases without any apparent degradation of the protein moiety of the molecule. The fact that precipitating and neutralizing antisera against factor VIII were obtained, shows that the molecules modified by EDTA and thrombin still have the antigenic properties of factor VIII. The inhibitor developed by hemophiliacs transfused with human factor VIII is bound to bovine factor VIII, forming a complex reveal that it is composed of bovine factor VIII and human gamma-globulin. Bovine factor VIII in the complex retains some antigenic determinants which bind rabbit antiserum against bovine factor VIII, as shown by neutralization of the antiserum and by precipitation studies.

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.

Purification and isolation of certain intermediates formed prior to the activation of prothrombin.

ArticlePurification and Isolation of Certain Intermediates Formed Prior to the Activation of ProthrombinJoseph E. Flynn, and Robert W. CoonJoseph E. FlynnFrom the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York City, and Robert W. CoonFrom the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York CityPublished Online:31 Oct 1953https://doi.org/10.1152/ajplegacy.1953.175.2.289MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByCoagulation of the Blood22 November 2006Membrane-dependent reactions in blood coagulation: role of the vitamin K-dependent enzyme complexesBiochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Vol. 1227, No. 3Activation of bovine factor VII (proconvertin) by factor XIIa (activated Hageman factor)1 May 2002 | Biochemistry, Vol. 16, No. 19Mechanism of activation of bovine factor IX (Christmas factor) by bovine factor XIa(activated plasma thromboplastin antecedent)1 May 2002 | Biochemistry, Vol. 13, No. 22Simultaneous Purification of Bovine Prothrombin and Factor XJournal of Biological Chemistry, Vol. 248, No. 22Bovine factor X 1 (Stuart factor). Mechanism of activation by a protein from Russell's viper venom1 May 2002 | Biochemistry, Vol. 11, No. 26Physiology and Biochemistry of Blood CoagulationREFERENCESActa Physiologica Scandinavica, Vol. 75Application of A Generalized Rate Equation to the Gaseous Reduction of Iron Oxide18 July 2013 | Canadian Metallurgical Quarterly, Vol. 5, No. 4The Kinetics of Hematite Reduction by Partially Reformed Natural Gas18 July 2013 | Canadian Metallurgical Quarterly, Vol. 5, No. 3The Proteins of Blood CoagulationPurification and Properties of Bovine Factor X: Molecular Changes During Activation *1 May 2002 | Biochemistry, Vol. 3, No. 12Congenital deficiency of factor VII (hypoconvertinemia)The American Journal of Medicine, Vol. 37, No. 1The Activity of Lung Microsomes in Blood CoagulationJournal of Biological Chemistry, Vol. 239, No. 3Coagulation, Hemorrhage and ThrombosisNew England Journal of Medicine, Vol. 252, No. 12[16] Enzymes in blood clotting More from this issue > Volume 175Issue 2October 1953Pages 289-295 Copyright & PermissionsCopyright © 1953 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1953.175.2.289PubMed13114399History Received 29 June 1953 Published online 31 October 1953 Published in print 31 October 1953 Metrics