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Cellular activation of factor IX (christmas factor)
THROMBOSIS @Pergamon
RESEARCH Vol. 13 pp. jOl-507 Press Ltd. 1978. Printed in Great Britain 00~~-3848/78/0~01-0j01
$02.00/o
CELLULAR ACTIVATION OF FACTOR IX (CHRISTMAS FACTOF.)l Henry S. Kingdon, John C. Herion and P. Gregory Rausch Departments of Medicine and aiochemistry Research Center and the Dental University of North Carolina Chapel Hill, North Carolina 27514 U.S.A.
(Received
3.7.1978.
Accepted
by
Editor
L.
Lorand)
ABSTRACT We have demonstrated Factor IX activation by sonicated polymorphonuclear leukocytes (PMNs). This activation reaction required the disrupted leukocytes, calcium chloride, and a small amount of normal human plasma. The requirement for normal plasma was met by plasma deficient in all of the known coagulation factors, and thus the substance present in normal plasma which facilitates this reaction was not identified. The fact that factor XI deficient plasma supported the reaction as well as normal plasma implied that factor XIa was not involved in this activation. Strontium ions could not substitute for calcium ions in the activation reaction, also implying that factor XIa was not involved. This factor IX activating principle in leukocytes could provide a mechanism for by-passing the contact factors of blood coagulation, thus providing an explanation for the discrepancy in clinical severity between deficiencies of the contact factors on the one hand and hemophilias A and B on the other. INTRODUCTION A central feature of the cascade hypothesis of blood clotting protein interactions is that plasma proenzymes are converted to enzymatically active forms in a stepwise fashion by previously formed active enzymes (1). Factor XIa is thought to arise by the action of Factors XIIa, Fletcher Factor, and Fitzgerald Factor upon Factor XI (2). Factor XIa in turn catalyzes the conversion of Factor IX to Factor IXa in the presence of calcium ion (3-5). This formulation of the early steps of intrinsic blood coagulation causes a dilemma: how is it that hemophilia A (Factor VIII deficiency) and hemophilia B (Factor IX deficiency) are such serious
1Kesearch Supported by grants HL-16633, DE-02G68, HL-17835, and XL-07149 from the iiational Institutes of Ucalth. 501
502
CELLULAR
ACTIVATION
OF FACTOR
IX
Vol.l3,No.3
inherited bleeding disorders,while deficiency of Factor XI leads only to a mild bleeding disorder, and deficienciesof Factor XII, Fletcher Factor and FitzgeraldFactor lead to no bleeding disorder whatsoever? Complex hypothesescould be proposed to answer this question but the simplest explanationwould be an alternativepathway for the activationof Factor IX. The purpose of this communicationis to demonstrate the presence of such a pathway involvingpolymorphonuclearleukocytes. We chose these cells because prior research had demonstratedthat they contain a procoagulant (6). MATERIALS AND METHODS were prepared from whole human blood Polymorphonuclearleukocytes following ammonium chloride lysis, by sedimentationon Ficoll-Hypaque gradients (7). The cells obtained were 98-100% pure as judged by phase contrast microscopy. After washing and suspending in normal saline, the cells were subjected to sonication in two 10 second bursts, and used without further purification. During most of these experiments the Factor IX substratewas the bovine barium sulfate eluate treated with phenylmethylsulfonylfluoride described previously in our laboratories (8)‘ but in one instance a human Factor IX concentratewas also used (9).
Factor IX substratewas incubated at 37OC with cell sonicate and CaC12. This mixture is referred to subsequentlyas the activationmixture. At indicated time intervals,aliquots were removed and diluted in ice-cold buffer; the dilution and the reduction in temperatureeffectivelystop the activation reaction. Aliquots of these diluted samples were then assayed in duplicate in a non-activatedpartial thromboplastintime system (HAPTT) as described previously (10). Progressive shortening of the MAPTT over the course of the experimentwas taken to indicate Factor IX activation. RESULTS The results of a Factor IX activation experiment are shown in Table I. Sonicated polymorphonuclearleukocytes activated Factor IX in the presence of normal plasma and calcium chloride. Cell sonicate, plasma, and calcium chloride are all required for the reaction.
TABLE I
Activation of Factor IX by Cell Sonicates I
Minutes of Incubation
0
ZlottinqTime i Minus Cell Sonicate
Seconds Minus Plasma
Minus CaC12
137
141
139
125
Complege System
5
128
139
140
130
10
105
137
136
147
15
84
132
138
156
aComplete system contains 0.3 ml Factor IX substrate (phenylmethylsulfonyl
Vol.l3,No.3
CELLKLAR
ACTIV.~TIO~
OF FACTOR
IX
fluoridg-treated BaSO eluate from bovine serum (E)), 0.1 ml cell sonicate 13 :< 10 P?DI/ml norma 4 saline, 10 ~1 normal human plasma: reaction started b:raddition of 0.1 ml .025 :!CaC12. 0.1 ml samples removed at indicated time intervais, diluted in 0.9 ml ice cold buffer (.06 M tris hydroxyinethylaminomethane - .09 M NaCl, pH 7.5). 0.1 ml aliquots of these diluted samples were then assayed in duplicate in an NAPTT (non-activated partial thromboplastin time) assay using non-contact activated human plasma as substrate (lo), and the time taken for clotting recorded in seconds. The standard NAPTT contained non-contact activated human plasma as a substrate. When deficient plasmas were substituted for normal plasma in the NAPTT, results were obtained as shown in Table II.
TABLE II Assay of PMN-Activated Factor IX With Various Substrate Plasmas Clotting Time in Seconds Minutes of Incubation
Normal Plasmaa
Plasma Used in NAPTT Assay IX Deficignt VIII Deficient Plasma Plasma
0
137
190
700
15
84
95
610
aActivation mixture and assays as described in Table I. L
UA~
in a, but with indicated deficient plasma substituted for normal plasma in NAPTT assay.
Activation was observed when Factor IX deficient plasma was used in place of normal plasma, as would be expected if the active enzyme evolved in the activation mixture were Factor IXa. However, no activation was detected when Factor VIII deficient plasma was substituted for normal plasma, indicating that negligible amounts of Factor Xa and thrombin were generated during the activation. We next attempted to identify the plasma factor present in the normal plasma included in the activation mixture by substituting deficient plasmas for this normal plasma. The results are shown in Tables III and IV. The two sets of data were obtained in similar experiments, except that for the experiment reported in Table III bovine barium sulfate eluate was used as substrate for the enzyme from PMNs, and for the experiment reported in Table IV the Factor IX substrate was a Factor IX concentrate prepared from human plasma.
CELLULAR
504
ACTIVATION
OF FACTOR
IX
Vo1.13,No.3
TABLE III
Activation of Factor IX by Sonicated PMNs In the Presence of Normal and Deficient Plasmas
Clotting Time in Seconds Ninutes of Plasma Included in Reaction Mixture Incubation b b b b Normala XI-Def. IX-Def.b VIII-Def.b X-Def. VII-Def. V-Def. / 0
157
155
144
159
150
153
155
15
81
107
100
104
110
101
105
aActivationmixture and assays as described in Table I. b As in a, but with indicated deficient plasma substituted for normal plasma in activationmixture.
It can be seen that plasmas deficient in all of the known coagulation factors supported the activation of Factor IX by cell sonicates, as did plasma depleted by plasminogen by passage through lysine-agarose (11). In experimentsnot shown here plasma heated to 56oC for 30 minutes also supported the reaction.
TABLE IV Activation of Factor IX by Sonicated PMNs In the Presence of Normal and Deficient Plasmasa
Clotting Time in Seconds Plasma Included in Reaction Mixture Minutes of Incubation Normal XII- Fletcher Fitzgerald XIII- Plasminogen Normal Def. Def. Def. Def. Depleted 0
221
211
260
285
270
230
310
30
90
119
141
125
76
105
120
aAs in Table III, but with human Factor IX substrate (9) substituted for bovine Factor IX substrate (8).
Thus, we can conclude that the requirement for normal plasma is not for one of the known coagulationproteins or for complement. These experiments did not establish the identity of this plasma cofactor.
CELLLXAR
vo1.13,?io.3
ACTIVATION
OF F.lCTOR IX
505
It was important to establish that s'actor Z:Ia was not involved i:]the activation of Factor IX, and that we were dealing with an entity operating independent of Factor XIa. We have shown previously that strontium ions can substitute for calcium ions in the activation of Factor IX by Factor XIa (12), .with a reaction rate in the presence of strontium about 70-80% of the rate in the presence of calcium. Strontium ion was therefore substituted for calcium ion in the activation of Factor IX by cell sonicates. The result are shwon in Table V.
TABLE
V
Calcium Requirements for PMN Activation of Factor IX
Clotting Time in Seconds
:linutes of Incubation
Activation By PPINSonicate Substitute Sr++ Complete for Ca++ Systema
Activation By Factor XI Compleke SubsFitute SK++ System for Ca++
0
116
133
125
127
5
117
130
90
100
10
103
127
69
74
15
88
120
64
68
aAs in Table I. b As in a, but substituting bovine Factor XIa(8) (0.3 mg/ml) for cell sonicate.
Although strontium can support the activation of Factor IX by Factor XIa, it does not support the activation by cell sonicates. Therefore, we conclude that Factor XIa is not involved in the activation being observed with the cell sonicates.
DISCUSSION In previous experiments we have shown that Factor XIa evolved during clotting without the participation of cellular elements (13). Furthermore, in the case of bovine Factor XIa, the enzyme isolated from serum appeared to arise from the precursor proenzyme Factor XI (14). In contrast, the experiments reported here indicate that PMNs serve as a source of Factor IX activator, which acts in the absence of the known contact factors of blood coagulation. Other laboratories have reported Factor IX activators which by-pass the contact factors of blood coagulation (E-18). This is tha first report of Factor IX activation by blood cells. The presence of Factor IX activators in the polymorphonuclear leukocyte provides an
506
CELLUJaAR ACTIVATION
OF FACTOR
IX
Vol.l3,No.3
alternativemechanism for the activationof Factor IX. Cellular activation Of Factor IX could explain the discrepanciesin clinical severity between deficienciesof the contact factors on the one hand, and hemophiliasA and B on the other. Further investigationwill be needed to determine which mechanism for Factor IX activation is quantitativelymore significantin whole blood.
ACKNOWLEDGEMENT The authors thank Drs. Roger Lundblad, Gilbert C. White, II, and Richard I. Walker for valuable discussions and suggestions,Ms. Barbara Herbert for technical assistance,and Ms. Wanda Dodson for secretarial assistance. We also thank Dr. John G. Watt for the generous gift of human Factor IX concentrateused in some of the experiments.
REFERENCES 1.
Davie, E.W. and Fujikawa, K. Basic Mechanisms in Blood Coagulation. Ann. Rev. Biochem. 44, 799, 1975.
2. Bouma, B.N. and Griffin, J.H. Human Blood CoagulationFactor XI. Purification,Properties,and Mechanism of Activation by Activated Factor XII. J. Biol. Chem. 252, 6432, 1977. 3.
Ratnoff, O.D. and Davie, E.W. The Activation of Christmas Factor (Factor IX) by Activated Plasma ThromboplastinAntecedent (Activated Factor XI). Biochemistry.1, 677, 1962.
4.
Kingdon, H.S., Davie, E.W. and Ratnoff, O.D. The Reaction between Activated Plasma ThromboplastinAntecedent and Diisopropylphosphofluoridate.Biochemistry.3, 166, 1964.
5.
Kingdon, H.S. Evolution of Enzyme Activities and Fibrin Formation after Contact of Plasma with Glass. J. Biomed. Mater. Res. 3, 25, 1969.
6.
Saba, H.I., Herion, J.C., Walker, R.I. and Roberts, H.R. The ProcoagulantActivity of Granulocytes. Proc. Sot. Exp. Biol. Med. 142, 614, 1973.
7.
English, D. and Andersen, B.R. Single-StepSeparation of Red Blood Cells, Granulocytes,and Mononuclear Leukocyteson Discontinuous Density Gradients of Ficoll-Hypaque. J. Immunol. Methods. 5, 249, 1974.
8.
Lundblad, R.L. and Kingdon, H.S. Biochemistryof the Interactionsof Bovine Factors XIa and IX. Thromb. Diath. Haemorrh. Suppl. 57, 315, 1974.
9.
Middleton, S.M., Bennet, I.H. and Smith, J.K. A TherapeuticConcentrate of CoagulationFactors II, IX and X from Citrated, Factor VIII-Depleted Plasma. VOX Sang. 24, 441, 1973.
10. Kingdon, H.S., Lundblad, R.L., Veltkamp, J.J. and Aronson, D.L. PotentiallyThrombogenicMaterials in Factor IX Concentrates. Thromb.
Vo1.13,No.3
CELLLLAR
Diath. Haemorrh.
ACTIVATION
OF
FACTOR
IX
507
33, 617, 1975.
11.
Deutsch, D.G. and Mertz, E.T. Plasminogen: Purification from Human Plasma by Affinity Chromatography. Science. 170, 1095, 1970.
12.
Kingdon, H.S. and Davie, E.W. Further Studies on the Activation of Factor IX by Activated Factor XI. Thromb. Diath. Haemorrh. Suppl. 17, 15, 1965.
13.
Kingdon, H.S. and Lundblad, R.L. Factors Affecting the Evolution of Factor XIa During Blood Coagulation. J. Lab. Clin. Med. 85, 826, 1975.
14.
White II, G.C., Kociba, G.J. and Kingdon, H.S. Factor IX Activator Content of Normal and Factor XI Deficient Serum. Thrombosis Research, in press.
15.
Irwin, J.F. and Mammen, E.F. Activation of Factor IX by Thrombin Preparations in the Absence of Factor XI. Thromb. Res. 8, 141, 1976.
16.
@sterud, B., Laake, K. and Prydz, H. The Activation of Human Factor IX. Thromb. Diath. Haemorrh. 33, 553, 1975.
17.
Kalousek, F., Konigsberg, W. and Nemerson, Y. Activation of Factor IX by Activated Factor X: A Link Between the Extrinsic and Intrinsic Coagulation Systems. FEBS Lett. 50, 382, 1975.
13.
Qsterud, B. and Rapaport, S.I. Activation of Factor IX by the Reaction Product of Tissue Factor and Factor VII: Additional Pathway for Initiating Blood Coagulation. Proc. Natl. Acad. Sci. USA. 74, 5260, 1077.
RESEARCH Vol. 13 pp. jOl-507 Press Ltd. 1978. Printed in Great Britain 00~~-3848/78/0~01-0j01
$02.00/o
CELLULAR ACTIVATION OF FACTOR IX (CHRISTMAS FACTOF.)l Henry S. Kingdon, John C. Herion and P. Gregory Rausch Departments of Medicine and aiochemistry Research Center and the Dental University of North Carolina Chapel Hill, North Carolina 27514 U.S.A.
(Received
3.7.1978.
Accepted
by
Editor
L.
Lorand)
ABSTRACT We have demonstrated Factor IX activation by sonicated polymorphonuclear leukocytes (PMNs). This activation reaction required the disrupted leukocytes, calcium chloride, and a small amount of normal human plasma. The requirement for normal plasma was met by plasma deficient in all of the known coagulation factors, and thus the substance present in normal plasma which facilitates this reaction was not identified. The fact that factor XI deficient plasma supported the reaction as well as normal plasma implied that factor XIa was not involved in this activation. Strontium ions could not substitute for calcium ions in the activation reaction, also implying that factor XIa was not involved. This factor IX activating principle in leukocytes could provide a mechanism for by-passing the contact factors of blood coagulation, thus providing an explanation for the discrepancy in clinical severity between deficiencies of the contact factors on the one hand and hemophilias A and B on the other. INTRODUCTION A central feature of the cascade hypothesis of blood clotting protein interactions is that plasma proenzymes are converted to enzymatically active forms in a stepwise fashion by previously formed active enzymes (1). Factor XIa is thought to arise by the action of Factors XIIa, Fletcher Factor, and Fitzgerald Factor upon Factor XI (2). Factor XIa in turn catalyzes the conversion of Factor IX to Factor IXa in the presence of calcium ion (3-5). This formulation of the early steps of intrinsic blood coagulation causes a dilemma: how is it that hemophilia A (Factor VIII deficiency) and hemophilia B (Factor IX deficiency) are such serious
1Kesearch Supported by grants HL-16633, DE-02G68, HL-17835, and XL-07149 from the iiational Institutes of Ucalth. 501
502
CELLULAR
ACTIVATION
OF FACTOR
IX
Vol.l3,No.3
inherited bleeding disorders,while deficiency of Factor XI leads only to a mild bleeding disorder, and deficienciesof Factor XII, Fletcher Factor and FitzgeraldFactor lead to no bleeding disorder whatsoever? Complex hypothesescould be proposed to answer this question but the simplest explanationwould be an alternativepathway for the activationof Factor IX. The purpose of this communicationis to demonstrate the presence of such a pathway involvingpolymorphonuclearleukocytes. We chose these cells because prior research had demonstratedthat they contain a procoagulant (6). MATERIALS AND METHODS were prepared from whole human blood Polymorphonuclearleukocytes following ammonium chloride lysis, by sedimentationon Ficoll-Hypaque gradients (7). The cells obtained were 98-100% pure as judged by phase contrast microscopy. After washing and suspending in normal saline, the cells were subjected to sonication in two 10 second bursts, and used without further purification. During most of these experiments the Factor IX substratewas the bovine barium sulfate eluate treated with phenylmethylsulfonylfluoride described previously in our laboratories (8)‘ but in one instance a human Factor IX concentratewas also used (9).
Factor IX substratewas incubated at 37OC with cell sonicate and CaC12. This mixture is referred to subsequentlyas the activationmixture. At indicated time intervals,aliquots were removed and diluted in ice-cold buffer; the dilution and the reduction in temperatureeffectivelystop the activation reaction. Aliquots of these diluted samples were then assayed in duplicate in a non-activatedpartial thromboplastintime system (HAPTT) as described previously (10). Progressive shortening of the MAPTT over the course of the experimentwas taken to indicate Factor IX activation. RESULTS The results of a Factor IX activation experiment are shown in Table I. Sonicated polymorphonuclearleukocytes activated Factor IX in the presence of normal plasma and calcium chloride. Cell sonicate, plasma, and calcium chloride are all required for the reaction.
TABLE I
Activation of Factor IX by Cell Sonicates I
Minutes of Incubation
0
ZlottinqTime i Minus Cell Sonicate
Seconds Minus Plasma
Minus CaC12
137
141
139
125
Complege System
5
128
139
140
130
10
105
137
136
147
15
84
132
138
156
aComplete system contains 0.3 ml Factor IX substrate (phenylmethylsulfonyl
Vol.l3,No.3
CELLKLAR
ACTIV.~TIO~
OF FACTOR
IX
fluoridg-treated BaSO eluate from bovine serum (E)), 0.1 ml cell sonicate 13 :< 10 P?DI/ml norma 4 saline, 10 ~1 normal human plasma: reaction started b:raddition of 0.1 ml .025 :!CaC12. 0.1 ml samples removed at indicated time intervais, diluted in 0.9 ml ice cold buffer (.06 M tris hydroxyinethylaminomethane - .09 M NaCl, pH 7.5). 0.1 ml aliquots of these diluted samples were then assayed in duplicate in an NAPTT (non-activated partial thromboplastin time) assay using non-contact activated human plasma as substrate (lo), and the time taken for clotting recorded in seconds. The standard NAPTT contained non-contact activated human plasma as a substrate. When deficient plasmas were substituted for normal plasma in the NAPTT, results were obtained as shown in Table II.
TABLE II Assay of PMN-Activated Factor IX With Various Substrate Plasmas Clotting Time in Seconds Minutes of Incubation
Normal Plasmaa
Plasma Used in NAPTT Assay IX Deficignt VIII Deficient Plasma Plasma
0
137
190
700
15
84
95
610
aActivation mixture and assays as described in Table I. L
UA~
in a, but with indicated deficient plasma substituted for normal plasma in NAPTT assay.
Activation was observed when Factor IX deficient plasma was used in place of normal plasma, as would be expected if the active enzyme evolved in the activation mixture were Factor IXa. However, no activation was detected when Factor VIII deficient plasma was substituted for normal plasma, indicating that negligible amounts of Factor Xa and thrombin were generated during the activation. We next attempted to identify the plasma factor present in the normal plasma included in the activation mixture by substituting deficient plasmas for this normal plasma. The results are shown in Tables III and IV. The two sets of data were obtained in similar experiments, except that for the experiment reported in Table III bovine barium sulfate eluate was used as substrate for the enzyme from PMNs, and for the experiment reported in Table IV the Factor IX substrate was a Factor IX concentrate prepared from human plasma.
CELLULAR
504
ACTIVATION
OF FACTOR
IX
Vo1.13,No.3
TABLE III
Activation of Factor IX by Sonicated PMNs In the Presence of Normal and Deficient Plasmas
Clotting Time in Seconds Ninutes of Plasma Included in Reaction Mixture Incubation b b b b Normala XI-Def. IX-Def.b VIII-Def.b X-Def. VII-Def. V-Def. / 0
157
155
144
159
150
153
155
15
81
107
100
104
110
101
105
aActivationmixture and assays as described in Table I. b As in a, but with indicated deficient plasma substituted for normal plasma in activationmixture.
It can be seen that plasmas deficient in all of the known coagulation factors supported the activation of Factor IX by cell sonicates, as did plasma depleted by plasminogen by passage through lysine-agarose (11). In experimentsnot shown here plasma heated to 56oC for 30 minutes also supported the reaction.
TABLE IV Activation of Factor IX by Sonicated PMNs In the Presence of Normal and Deficient Plasmasa
Clotting Time in Seconds Plasma Included in Reaction Mixture Minutes of Incubation Normal XII- Fletcher Fitzgerald XIII- Plasminogen Normal Def. Def. Def. Def. Depleted 0
221
211
260
285
270
230
310
30
90
119
141
125
76
105
120
aAs in Table III, but with human Factor IX substrate (9) substituted for bovine Factor IX substrate (8).
Thus, we can conclude that the requirement for normal plasma is not for one of the known coagulationproteins or for complement. These experiments did not establish the identity of this plasma cofactor.
CELLLXAR
vo1.13,?io.3
ACTIVATION
OF F.lCTOR IX
505
It was important to establish that s'actor Z:Ia was not involved i:]the activation of Factor IX, and that we were dealing with an entity operating independent of Factor XIa. We have shown previously that strontium ions can substitute for calcium ions in the activation of Factor IX by Factor XIa (12), .with a reaction rate in the presence of strontium about 70-80% of the rate in the presence of calcium. Strontium ion was therefore substituted for calcium ion in the activation of Factor IX by cell sonicates. The result are shwon in Table V.
TABLE
V
Calcium Requirements for PMN Activation of Factor IX
Clotting Time in Seconds
:linutes of Incubation
Activation By PPINSonicate Substitute Sr++ Complete for Ca++ Systema
Activation By Factor XI Compleke SubsFitute SK++ System for Ca++
0
116
133
125
127
5
117
130
90
100
10
103
127
69
74
15
88
120
64
68
aAs in Table I. b As in a, but substituting bovine Factor XIa(8) (0.3 mg/ml) for cell sonicate.
Although strontium can support the activation of Factor IX by Factor XIa, it does not support the activation by cell sonicates. Therefore, we conclude that Factor XIa is not involved in the activation being observed with the cell sonicates.
DISCUSSION In previous experiments we have shown that Factor XIa evolved during clotting without the participation of cellular elements (13). Furthermore, in the case of bovine Factor XIa, the enzyme isolated from serum appeared to arise from the precursor proenzyme Factor XI (14). In contrast, the experiments reported here indicate that PMNs serve as a source of Factor IX activator, which acts in the absence of the known contact factors of blood coagulation. Other laboratories have reported Factor IX activators which by-pass the contact factors of blood coagulation (E-18). This is tha first report of Factor IX activation by blood cells. The presence of Factor IX activators in the polymorphonuclear leukocyte provides an
506
CELLUJaAR ACTIVATION
OF FACTOR
IX
Vol.l3,No.3
alternativemechanism for the activationof Factor IX. Cellular activation Of Factor IX could explain the discrepanciesin clinical severity between deficienciesof the contact factors on the one hand, and hemophiliasA and B on the other. Further investigationwill be needed to determine which mechanism for Factor IX activation is quantitativelymore significantin whole blood.
ACKNOWLEDGEMENT The authors thank Drs. Roger Lundblad, Gilbert C. White, II, and Richard I. Walker for valuable discussions and suggestions,Ms. Barbara Herbert for technical assistance,and Ms. Wanda Dodson for secretarial assistance. We also thank Dr. John G. Watt for the generous gift of human Factor IX concentrateused in some of the experiments.
REFERENCES 1.
Davie, E.W. and Fujikawa, K. Basic Mechanisms in Blood Coagulation. Ann. Rev. Biochem. 44, 799, 1975.
2. Bouma, B.N. and Griffin, J.H. Human Blood CoagulationFactor XI. Purification,Properties,and Mechanism of Activation by Activated Factor XII. J. Biol. Chem. 252, 6432, 1977. 3.
Ratnoff, O.D. and Davie, E.W. The Activation of Christmas Factor (Factor IX) by Activated Plasma ThromboplastinAntecedent (Activated Factor XI). Biochemistry.1, 677, 1962.
4.
Kingdon, H.S., Davie, E.W. and Ratnoff, O.D. The Reaction between Activated Plasma ThromboplastinAntecedent and Diisopropylphosphofluoridate.Biochemistry.3, 166, 1964.
5.
Kingdon, H.S. Evolution of Enzyme Activities and Fibrin Formation after Contact of Plasma with Glass. J. Biomed. Mater. Res. 3, 25, 1969.
6.
Saba, H.I., Herion, J.C., Walker, R.I. and Roberts, H.R. The ProcoagulantActivity of Granulocytes. Proc. Sot. Exp. Biol. Med. 142, 614, 1973.
7.
English, D. and Andersen, B.R. Single-StepSeparation of Red Blood Cells, Granulocytes,and Mononuclear Leukocyteson Discontinuous Density Gradients of Ficoll-Hypaque. J. Immunol. Methods. 5, 249, 1974.
8.
Lundblad, R.L. and Kingdon, H.S. Biochemistryof the Interactionsof Bovine Factors XIa and IX. Thromb. Diath. Haemorrh. Suppl. 57, 315, 1974.
9.
Middleton, S.M., Bennet, I.H. and Smith, J.K. A TherapeuticConcentrate of CoagulationFactors II, IX and X from Citrated, Factor VIII-Depleted Plasma. VOX Sang. 24, 441, 1973.
10. Kingdon, H.S., Lundblad, R.L., Veltkamp, J.J. and Aronson, D.L. PotentiallyThrombogenicMaterials in Factor IX Concentrates. Thromb.
Vo1.13,No.3
CELLLLAR
Diath. Haemorrh.
ACTIVATION
OF
FACTOR
IX
507
33, 617, 1975.
11.
Deutsch, D.G. and Mertz, E.T. Plasminogen: Purification from Human Plasma by Affinity Chromatography. Science. 170, 1095, 1970.
12.
Kingdon, H.S. and Davie, E.W. Further Studies on the Activation of Factor IX by Activated Factor XI. Thromb. Diath. Haemorrh. Suppl. 17, 15, 1965.
13.
Kingdon, H.S. and Lundblad, R.L. Factors Affecting the Evolution of Factor XIa During Blood Coagulation. J. Lab. Clin. Med. 85, 826, 1975.
14.
White II, G.C., Kociba, G.J. and Kingdon, H.S. Factor IX Activator Content of Normal and Factor XI Deficient Serum. Thrombosis Research, in press.
15.
Irwin, J.F. and Mammen, E.F. Activation of Factor IX by Thrombin Preparations in the Absence of Factor XI. Thromb. Res. 8, 141, 1976.
16.
@sterud, B., Laake, K. and Prydz, H. The Activation of Human Factor IX. Thromb. Diath. Haemorrh. 33, 553, 1975.
17.
Kalousek, F., Konigsberg, W. and Nemerson, Y. Activation of Factor IX by Activated Factor X: A Link Between the Extrinsic and Intrinsic Coagulation Systems. FEBS Lett. 50, 382, 1975.
13.
Qsterud, B. and Rapaport, S.I. Activation of Factor IX by the Reaction Product of Tissue Factor and Factor VII: Additional Pathway for Initiating Blood Coagulation. Proc. Natl. Acad. Sci. USA. 74, 5260, 1077.