Differential Consumption of Coagulation Factors Resulting from Activation of the Extrinsic (Tissue Thromboplastin) or the Intrinsic (Foreign Surface Contact) Pathways

1. The consumption of coagulation Factors IX, X, and XI was studied innormal whole blood clotted with celite (intrinsic activation) or tissue thromboplastin (extrinsic activation).

2. During these studies an assay method for Factor XI was developed whichwas not influenced by the presence of tissue thromboplastin. The assay methodis based on the thromboplastin generation principle.

3. When blood clotted in silicone treated tubes, the serum and plasma concentrations of Factors IX, X, and XI were almost identical, indicating that littleconsumption or activation of these factors had occurred.

4. In the presence of celite, coagulation Factors IX and XI are consumed,whereas Factor X is consumed only slightly.

5. In the presence of tissue thromboplastin, Factor X is consumed, whereasFactors IX and XI are not consumed.

6. In the presence of both celite and thromboplastin, the thromboplastindecreased the consumption of Factors IX and XI produced by celite.

7. The study of serum coagulation factor levels may provide evidence as towhether the coagulation process had been initiated by the intrinsic (foreignsurface contact) or extrinsic (thromboplastin) pathways.

Submitted on May 9, 1966 Accepted on July 21, 1966     

Congenital Haemorrhagic Condition Similar but Not Identical to Factor X Deficiency

A 23-year-old white male with a bleeding tendency since early childhood presented a congenital coagulation defect similar but not identical to factor X deficiency. A first and second stage defect were demonstrated, characterized by a prolonged prothrombin time, prolonged partial thromboplastin time, abnormal thromboplastin generation, abnormal prothrombin consumption. The Stypven clotting time was slightly prolonged on fresh plasma but was normal on frozen plasma. Factors I, II, V, VIII, IX, XI, and XII were all within normal limits; factor VII was at the lower limits of normally or slightly decreased. Mr. Stuart's plasma failed to correct the defect of the patients plasma; however, a known factor VII deficient plasma was able to correct the abnormality. Factor X levels showed low (3–13%) only when assayed using tissue whole thromboplastin or tissue partial thromboplastin; the factor X assay using a Stypven-cephalin mixture yielded normal or near normal values. The factor II + factor X level using a Stypven-cephalin mixture appeared normal also. The significance of the findings is discussed. The results are tentatively interpreted as being due to an abnormal factor X rather than to a real deficiency.     

Do Platelets Enhance Thromboplastin —Induced Intravascular Clotting?

During intravenous infusion of homologous tissue thromboplastin in normal rabbits, nearly 60% of the platelets initially present disappeared, but this platelet injury and destruction did not enhance intravascular clotting. Thus, clotting was equally severe in normal animals and in animals made extremely thrombocytopenic (mean: 4,000/,μ1, range: 1,000–8,000/μl) by the alkylating agent busulphan; more than half of the circulating fibrinogen and about two thirds of factor V activity were consumed, accompanied by significant decreases in the levels of factors VII and IX. Haemolysis was observed secondary to clotting, but never the generalized Shwartzman reaction.     

A "new" congenital haemorrhagic condition due to the presence of an abnormal factor X (factor X Friuli): study of a large kindred

Summary Three related patients are presented who show a congenital coagulation disorder with laboratory features intermediate between classical factor-VII and factor-X deficiencies. A woman and two men had suffered from bleeding since early childhood, with epistaxis, bleeding from the gums, post-traumatic haemarthroses, bleeding after tooth extractions and other surgical procedures. Investigation demonstrated a prolonged prothrombin time, prolonged partial thromboplastin time, abnormal prothrombin consumption and abnormal thromboplastin generation corrected by normal serum. Platelet and vascular tests were normal and no hyperfibrinolysis was found. Factors I, II, V, VII, IX, XI and XII were within normal limits in all three patients. Mutual correction was demonstrated with a known factor-VII-deficient plasma but not with Stuart (X-deficient) plasma. Factor-X assay yielded low (4–9%) levels using tissue whole thromboplastin or tissue partial thromboplastin; but the results were normal with a Stypven-cephalin mixture. In agreement with these results, the Stypven-cephalin clotting time, the Stypven clotting time and the factor II + factor X level using a Stypven-cephalin mixture were normal, ‘correction’ being attributable to the Russell's Viper venom. These results were thought to indicate an abnormal factor X rather than a real deficiency. The presence of abnormal factor X was demonstrated by the antibody neutralization technique and by the immunodiffusion studies. The defect, like classical factor X deficiency, is transmitted as an autosomal incompletely recessive trait. The heterozygote population has factor-X levels varying from 32% to 55% of normal and are usually asymptomatic. The term ‘Factor X Friuli’ is proposed for the abnormality, due to a locally common mutant gene.     

Activated Coagulation Factors: In-Vivo and in-Vitro Studies

In the course of serial coagulation studies on patients with acute leukaemia, defibrination was frequently encountered in the lymphoblastic, myeloblastic and promyelocytic varieties of the disease. Factor-VIII levels were often high and varied very considerably, a particular phenomenon being the association at times of very high one-stage levels with decreased two-stage levels. In one patient heparin infusion was associated with a rise in the two-stage Factor-VIII level and cessation of the infusion with a prompt drop in level. Less frequently, gross elevation of one-stage Factor-XI activity occurred compared with two-stage (eluate test) levels. In order to study possible mechanisms for the phenomena observed, the effect of adding various pro-coagulants to normal plasma on the one- and two-stage methods for measuring Factors VIII, IX and XI was studied. Thrombin caused a marked rise in one-stage Factor-VIII activity and a much smaller rise in one-stage Factor IX and XI measurements. Two-stage Factor-VIII activity was reduced and Factors IX and XI little affected. These findings simulate some of the findings in vivo. The effect of activated Factors IX, X and XI on the one-stage activity of Factors VIII, IX and XI may be explained by assuming that an activated product increases the one-stage activity of any factor which participates in the clotting reaction sequence at a stage preceding the formation of the product. The actual findings were that an activated Factor-X preparation increased one-stage Factors VIII, IX and XI measurements; an activated Factor-IX preparation considerably increased one-stage Factor IX and XI measurements but affected Factor-VIII activity only when used at very high concentrations; an activated Factor-XI preparation did not affect Factor VIII, IX or XI one-stage activity. None of these three activated products influenced the two-stage measurements of Factors VIII, IX and XI. Tissue thromboplastin increased one-stage Factor VIII, IX and XI activity to a similar extent, did not influence two-stage Factor VIII and IX measurements and slightly increased two-stage Factor-XI activity.     

Chromatographic Behaviour of Clotting Factors

Through the use of DEAE-cellulose under the conditions reported in this paper which were particularly selective for the adsorption of clotting factors, the chromatographic behaviour of Factors I, II, V, VII, VIII, IX and X was studied using different buffer systems. Human plasma, supernatant of Fraction I of Cohn and human Factor-VIII concentrates were used as starting materials. As some of the chromatographic systems used do not perceptibly modify the physical and chemical properties of the plasma or its derivatives, they allow the techniques to be included in a general scheme of routine plasma fractionation.
The conclusions drawn from the chromatographic behaviour of the factors studied have led to the preparation of a concentrate of Factors II, IX and X for clinical use, a concentrate of Factor VII and an artificial substrate for the assay of Factor VII.     

Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.     

The Coagulant Activity of Platelets

When platelet-rich plasma is incubated for 16–20 hours at 37° C. and the platelets are then separated and washed, tissue-factor activity develops in these platelets. The active platelets accellerate the clotting of plasma samples deficient in Factor XII, XI, IX or VIII, and of normal plasma; the coagulant activity for samples deficient in Factors V, VII and X is much less marked. The activity developed will cause activation of Factor X in a serum eluate, whereas no such activity is evident in platelets separated from plasma immediately after collection from the donor.
The development of tissue-factor activity of platelets depends on the presence of Factor XII but not on the presence of any of the other factors tested. The relationship of tissue-factor activity to platelet factor 3 is discussed.     

Activation of 125I-Factor IX and 125I-Factor X: Effect of Tissue Factor and Factor VII,Factor Xa and Thrombin

Activation of Factor IX and Factor X was studied by adding ¹²⁵I-Factor IX or ¹²⁵I-Factor X to reaction mixtures and quantitating cleavage products by reduced sodium dodecylsulfate gel electrophoresis. Thrombin failed to activate Factors IX or X; Factor X_a produced insignificant amounts of cleavage products of both factors. In contrast, the reaction product of tissue factor and Factor VII cleaved large amounts of both Factor IX and Factor X in purified systems and in plasma. In incubation mixtures of plasma containing added ¹²⁵I-Factor IX or ¹²⁵I-Factor X, tissue factor and Ca2+ions, the percentage of total radioactivity in the heavy chain peak of ¹²⁵I-IX_a and the heavy chain peak of ¹²⁵I-X_a increased at a similar rate. When the tissue factor was diluted, similar curves were obtained for percent cleavage of ¹²⁵I-Factor IX and percent cleavage of ¹²⁵I-Factor X plotted against tissue factor concentration. These findings support the hypothesis that activation of Facor IX by the tissue factor-Factor VII reaction product represents a physiologically significant step in normal haemostasis.     

Activation of human factor VII in the initiation of tissue factor- dependent coagulation

We have used activation peptide release assays to compare factor VII and activated factor VII (VIIa) activation of factor X, normal factor IX (IXN), and a variant factor IX (IXBmLE), which, after activation, is unable to back-activate factor VII. In purified systems, factor VII and VIIa each rapidly activated factor X, but after a one minute lag for factor VII. VIIa also readily activated both IXN and IXBmLE. Factor VII initially failed to activate substantial amounts of either IXN or IXBmLE; on further incubation factor VII activated IXN but not IXBmLE. Activation of IXN began when approximately 10% of factor VII had been converted to VIIa, as measured by 125I-factor VII radioactivity profiles. Adding factor VII to VIIa slowed its activation of IXBmLE. However, in the presence of factor X, factor VII alone rapidly activated IXBmLE. Unlike purified systems, 1 nmol/L VIIa added to factor VII-deficient plasma failed to activate factor IX. Increasing factor VII to 10 nmol/L (plasma concentration) either as native VII or VIIa yielded similar activation curves for factor IX and similar activation curves for factor X. Adding 5% VIIa to factor X-deficient plasma and to factor XII-deficient plasma substantially shortened the dilute tissue factor clotting time of only the former. These data support the hypothesis that factor VII/tissue factor complex initiates tissue factor-dependent clotting through a minimal generation of Xa. This Xa then rapidly back-activates a small amount of factor VII, following which the rates of activation of both factors IX and X increase dramatically.     

The inter-relationship of factor VII and its activity state with plasma lipids in healthy male adults

Summary A close inter-relationship between raised factor VII clotting activity and elevated blood lipids, particularly serum triglycerides, is well established. A study of factor VII, its activation state and of plasma lipids has been undertaken in two groups of healthy middle-aged males to elucidate this mechanism. A control group with normal factor VII levels were closely matched for age and body-mass index with a second group with elevated levels. Factor VII assays, using rabbit and bovine thromboplastin and a factor VII Ag method, were employed. Triglycerides correlated with the rabbit factor VII thromboplastin assay and factor VII Ag ( P <0.05) but not with the bovine thromboplastin method. Higher HDL-cholesterol and apolipoprotein A-I levels were found in subjects with increased factor VII ( P <0.001) and appeared to be due to differences in alcohol consumption. Cholesterol levels were significantly higher with elevated factor VII. Differential testing suggests that higher factor VII is predominantly mediated through a rise in total VII, rather than an increase in its activity state.     

Coagulopathy induced by continuous infusion of high doses of human lymphoblastoid interferon

Seven patients with myeloblastic leukemia were treated for 10 days with high-dose (15 or 30 million units/m2/day), human lymphoblastoid interferon (Wellferon) by continuous iv infusion. All patients developed prolonged activated partial thromboplastin time, and four developed prolonged prothrombin time. Factor assays demonstrated low levels of II, VII, IX, X, and XII. Coagulation abnormalities improved after discontinuation of interferon therapy.     

An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.     

Regulation of the procoagulant activity within the bronchoalveolar compartment of normal human lung

The nature of the procoagulant activity of normal bronchoalveolar fluid was examined both qualitatively and quantitatively. Unconcentrated, cell-free lavage freshly obtained from normal volunteers clotted normal plasma in a mean of 84 +/- 20 s. The procoagulant activity was initiated by Factor VII-tissue factor complexes as judged by differential activity in various plasmas genetically deficient in single clotting factors, by neutralization of the procoagulant activity with antibodies to either Factor VII or tissue factor, and by a Factor X activation assay. Preincubation of the lavage with calcium was required to demonstrate Factor VII activity in unconcentrated samples. The cell-free fluid contained about 8,500 thromboplastin units/mg protein, equivalent to a third of the thromboplastin standard and indicating high amounts of cofactor. Quantitation of Factor VII was estimated by functional analysis in coagulation and amidolytic assays with reference to dilutions of normal plasma of known Factor VII concentration. When lavage and diluted plasma were adjusted to yield equivalent amidolytic activities, the average ratio of the Factor VII-clotting activity of the alveolar fluid relative to plasma Factor VII was 19 +/- 7, suggesting the presence of Factor VIIa in lavage. In contrast to previous reports with serum or activated plasma, immunoblots of concentrated lavage revealed only single-chain Factor VII, and 125I-Factor VII added to the fluid was not converted to 125I-Factor VIIa, suggesting a unique control mechanism in the lung compartment which differs from plasma. When equivalent Factor VII amidolytic activities in diluted plasma and cell-free lavage were compared, the rates of Factor Xa formation were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coagulation factor activity in platelet concentrates stored up to 7 days: an in vitro and in vivo study

The in vitro and in vivo recovery of coagulation factor activity in platelet concentrates stored up to 172 h was studied. In vitro studies revealed that fibrinogen and antithrombin III levels do not change with storage. Factors II, V, VII, VIII, IX, X and XI all showed statistically significant falls from baseline over the 172 h storage period. However, most factor activities remained above 70%, with the major exception of factors V and VIII. These factors fell to less than 30% activity over the storage period, consistent with their known lability during whole blood storage. In vivo studies after platelet concentrate infusion in patients with concurrent thrombocytopenia and coagulation deficiencies revealed that the measured in vitro activity was recoverable in vivo. We conclude that platelet concentrates stored for 172 h are an adequate source of clotting factors. However, like stored whole blood, they may not provide therapeutic doses of factors V and VIII.     

Screening coagulation tests and clotting factors in homozygous beta-thalassemia.

In 30 children with homozygous beta-thalassemia the hemostasis screening tests (bleeding time, PT, PTT), platelet count and specific assays of clotting factors were carried out 25 days after their last transfusion. PT, PTT, and bleeding time showed minor variations; considerable thrombocytosis was found in splenectomized patients. Factors IX and XII were decreased in a high proportion of patients, the vitamin K-dependent factors (II, VII, IX, X) were slightly reduced and factors I, V and VIII remained within the normal range in a majority of patients. Hepatic failure resulting in defective protein synthesis does not explain the more marked impairment of factors XI and XII, which might be secondary to activation of the intrinsic coagulation and/or kallikrein systems following intravascular haemolysis and multiple blood transfusions.     

Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation.

A study was carried out on mechanisms, independent of activated Factor XI, capable of activating Factor IX. The reaction product of tissue factor and Factor VII functioned as a potent Factor IX activator in the assay system used. Activated Factor IX itself activated Factor X; thrombin failed to activate Factor IX. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis confirmed that the reaction product of tissue factor and Factor VII activated Factor IX, with replacement of the band corresponding to native factor IX [molecular weight (Mr) 55,000] by bands corresponding to the heavy chain (Mr 27,000) and light chain (Mr 17,000) of activated Factor IX. When either Factor VII or calcium ions were left out of incubation mixtures, the band of native Factor IX persisted unchanged. Contact of blood with tissue factor represents a second mechanism, bypassing activated Factor XI, for the activation of Factor IX during hemostasis. It may help to explain the discrepancy between the mild bleeding of hereditary Factor XI deficiency and the severe bleeding of hereditary Factor IX deficiency.     

Dietary fish lipids do not diminish platelet adhesion to subendothelium

There is a discrepancy in the results of reported studies of levels of vitamin K dependent coagulation factors in patients on warfarin therapy. This may have arisen partly because of the problem of assuring compliance with therapy in outpatients. The plasma concentrations of the vitamin K dependent clotting factors II, VII, IX and X were studied in 23 outpatients whose adherence to prescribed warfarin therapy was determined using a pharmacological indicator of compliance. In these patients, who were shown to have consistently good compliance and stable anticoagulant control over a period of 3-6 months, the activities in plasma of the four coagulation factors were not equally suppressed. Factor IX levels were significantly greater than those of factor VII (P less than 0.0001) which in turn were significantly greater than the levels of factor II (P less than 0.0001) or factor X (P less than 0.0001). There was no significant difference between the levels of factors II and X which were depressed to a similar extent. The proportion of variability of the International Normalized Ratio (INR) explained by linear regression was 51-77% and a model was derived to predict the INR from the mean of the levels of the four clotting factors. The concentrations of the coagulation factors II, VII, IX and X are likely to be highly dependent on the degree of compliance with warfarin therapy which should be taken into account when investigating the behaviour of these factors.     

A study of antihaemophilic globulin (factor VIII) consumption.

A study has been made of the rate of disappearance of factor VIII during the clotting of normal plasma samples and of samples deficient in factors V, VII, IX, X, XI and XII. The rate of disappearance (or consumption) of factor VIII was the same in the normal and factor VII deficient samples. Delayed disappearance of factor VIII was observed in samples deficient in factors V, IX, X, XI and XII. The significance of the findings to the theory of blood coagulation is discussed.     

Acquired factor X deficiency with associated defects in platelet aggregation: A response to corticosteroid therapy

A 79 year old white woman presented with a severe bleeding disorder. Evaluation revealed a prothrombin time of 27.6 seconds (control, 11 seconds) and an activated partial thromboplastin time of 61 seconds. Specific clotting factor assays showed an isolated deficiency of factor X ranging from 7 to 12 per cent on three determinations. Platelet aggregation and bleeding time were also abnormal in response to epinephrine and collagen. Factor X levels and platelet aggregation returned to normal and bleeding stopped after institution of corticosteroid therapy.