Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX- deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.     

Platelets and Initiation of Intrinsic Clotting

S ummary . Comparison of activities in platelet rich and platelet poor plasmas from normal donors and patients deficient in either factor VIII, IX, XI or XII indicates that platelets contain activities which can partially substitute for plasma factors XI and XII. The factor-XI-like activity is expressed in a one-stage activated partial thromboplastin assay and in an intact prothrombin consumption system. The factor-XII-like activity is scarcely detectable in a one-stage assay but markedly enhances the defective prothrombin consumption of factor XII deficient plasma. Intact prothrombin consumption tests with platelet poor plasmas fortified with cephalin show that in the presence of high concentrations of platelet factor 3 activity only trace contact activation is required to promote good prothrombin consumption. The platelet, by supplying both platelet factor 3 and activities bypassing plasma contact activation factors XI and XII, may provide an important route for activating intrinsic clotting.     

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.     

On the mechanism of inhibition of tissue factor pathway by the synthetic pentasaccharide during coagulation of human plasma.

The tissue factor (TF) pathway is preponderant in the initiation of blood coagulation in normal haemostasis and in thrombotic states. In the present study we investigated the mechanisms by which the synthetic pentasaccharide may influence the regulation of the TF pathway during clotting of human platelet poor plasma (PPP). Clotting of normal PPP or plasmas immuno-depleted of a single clotting factor (factor VII, factor XII, factor XI, factor IX, factor VIII, factor X, factor V, factor II) was initiated by triggering the TF pathway in the presence of fondaparinux (0.5 microg/ml). Activated factor VII (FVIIa) levels were measured in serum obtained at several time intervals after re-calcification of PPP. A clotting assay highly specific for FVIIa was used. The synthetic pentasaccharide inhibited the generation of FVIIa by 66%. The inhibitory effect of fondaparinux on FVIIa was completely abolished when antithrombin activity of plasma was inhibited by a specific antibody. Following the activation of TF pathway in plasmas depleted of factor X or factor IX, the inhibitory effect of fondaparinux on FVIIa generation was completely abolished, whereas it was not significantly modified when the other clotting factor-depleted plasmas were clotted. When fondaparinux was added in the serum, after the maximal generation of FVIIa, it inhibited by 20-30% the activity of the FVIIa-TF complex. These data suggest that fondaparinux enhances the antithrombin-dependent downregulation of the TF pathway by decreasing the generation of FVIIa via the inhibition of the generation and the activity of activated factor IX and activated factor X, and by inhibiting the activity of the FVIIa-TF complex.     

Importance of factor-IX-dependent prothrombinase formation--the Josso pathway--in clotting plasma

We report a study on the importance of factor IX activation in thromboplastin-dependent coagulation in plasma. Diluted, CaCl2-containing thromboplastin solutions at constant phospholipid concentration were used to trigger the coagulation in plasma from patients with congenital factor IX and factor VIII deficiency in the presence and absence of added factors IX and VIII, and the generation of thrombin activity was monitored. When coagulation is triggered with the high thromboplastin concentrations normally used in clinical routine tests, the generation of thrombin activity in plasma of patients with congenital factor IX deficiency before and after reconstitution with purified factor IX appears identical. When, however, coagulation is triggered with low thromboplastin concentrations, a clear dependency of the generation of thrombin activity on the concentration of factor IX becomes evident at factor IX concentrations lower than 30 nM (about 40% clotting factor activity). Factor VIII is a compulsory cofactor for this factor IX activity because the prothrombinase activity at optimal factor IX concentration is still critically dependent upon the amount of factor VIII present. The lower the amount of thromboplastin, the higher the importance of factor IX and factor VIII activation in thromboplastin-dependent coagulation. This suggests a role of this pathway in pathophysiological thrombin formation.     

A comparison of two APTT reagents which use silica activators

The Ortho activated partial thromboplastin time (APTT) reagent, Thrombosil 1 (TS), was compared to the General Diagnostics automated APTT reagent (GD). TS produced more precise results over a 38-day period of testing a normal control plasma, indicating that the upper limit of the normal range could be more precisely set with TS. This normal range was better represented if the normal values with both reagents were logarithmically transformed before calculating the mean +/- 2 SD. TS was more sensitive to plasma which had been heparinized in vitro. This was also demonstrated in vivo by the testing of 100 plasmas from heparinized patients. On testing of in-vitro dilutions of normal plasma with factor-deficient plasmas, TS was more sensitive to decreasing levels of factors VIII, IX and XI but less sensitive to decreasing factor XII. This was demonstrable in vivo in 71% of cases with plasmas from factor-deficient patients. GD was more sensitive to the lupus anticoagulant in most cases.     

Phospholipids accelerate factor IX activation by surface bound factor XIa

Activation of bovine factor IX by surface bound factor XIa which was generated either by activation of human citrated factor IX deficient plasma or a mixture of purified human factors XII, high molecular weight kininogen (HMWK) and XI in glass tubes, is accelerated by cephalin. Human brain cephalin in dilutions ranging from 1:5 to 1:500 was studied for its effect on the activation of factor IX in concentrations of 1.0 u/ml and 16 u/ml. Cephalin dilutions from 1:5 to 1:30 accelerated the activation of the concentrated factor IX sample two- to threefold. Protein cleavage of this factor IX sample in the presence of 1:30 cephalin occurred twice as fast as in the absence of cephalin. Activation of the dilute factor IX sample (1.0 u/ml) was most effectively accelerated by cephalin in dilutions from 1:30 to 1:250. In all experiments the presence of phospholipid led to an increased factor IX cleavage concomitantly with faster generation of factor IXa activity. The results demonstrate that phospholipids actively participate in blood coagulation at an earlier stage than previously described.     

Agarose gel method: its usefulness in assaying factor VIII inhibitors, evaluating treatment and suggesting a mechanism of action for factor IX concentrates

Plasmas from 14 patients with factor VIII inhibitors, 10 haemophiliacs and four non-haemophiliacs, were assayed by both the agarose gel and Bethesda methods. Good correlation was observed in 34 samples from 13 patients, but there was poor correlation in three samples from a single haemophilic patient. The sensitivity of the method was increased by diluting normal platelet rich plasma (PRP) with congenital factor VIII deficient plasma. With this modification, as little as 0.4 of a Bethesda unit (Bu) could be measured accurately. The agarose method is easier to perform and requires much less technician time than the Bethesda assay (Ba). Inhibitory activity can be measured even in the presence of large amounts of transfused factor VIII or factor IX concentrates. To study the effect of factor IX concentrates on the inhibitor, the method was modified by incorporating into the gels plasmas specifically deficient in either factor VII, IX or X. Our data suggest that factor VII is probably responsible for the 'bypassing' activity of factor IX concentrates.     

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.     

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     

Preparation, Characterization, and Activation of a Highly Purified Factor XI: Evidence that a Hitherto Unrecognized Plasma Activity Participates in the Interaction of Factors XI and XII

S ummary . Highly purified native factor XI has been prepared from normal plasma using a five step purification scheme. Purified factor XI is essentially free of factors II, V, VII, VIII, IX, X, XII, and Fletcher factor. No plasminogen-plasmin could be detected. Purified factor XI decays rapidly but can be stabilized with human serum albumin. Factor XI migrates between the β and γ globulins on starch block electrophoresis. It has an apparent molecular weight on gel filtration of 210 000. Purified factor XI is activated by weak trypsin. No detectable BAEe esterase activity accompanies this activation. Neither factor XII adsorbed to kaolin nor activated factor XII in solution could activate purified factor XI; both reagents activate factor XI in dilute factor XII deficient plasma. Hence, plasma appears to supply a third activity which facilitates the interaction of factors XI and XII. This activity is shown to be distinct from Fletcher factor.     

Influence of coagulation factors on intrinsic thrombin generation.

The intrinsic coagulation activity assay (INCA) is a new thrombin-generation test that imitates the intrinsic pathway of blood coagulation. The aim of the present study was to investigate the influence of the main coagulation factors on the INCA. The INCA was performed with citrated plasma samples supplemented with different amounts of fibrinogen. The INCA and activated partial thromboplastin time determination were performed with factor-depleted plasmas and with mixtures of depleted plasmas with normal plasma. Supplemented purified fibrinogen resulted in a decrease of intrinsic thrombin generation (50% inhibitory concentration = 0.8 g/l). The INCA depends on the intrinsic factors (factors VIII, IX, XI and XII) and on the factors of the common pathway (factors II, V and X): for normal thrombin generation, at least about 50% of normal factor II is necessary. For the majority of factors, the sensitivity of the INCA appears to be approximately one order of magnitude better than that of the activated partial thromboplastin time. The INCA allows one to diagnose defects in the intrinsic coagulation system and might be a useful test to support development and characterization of new drugs targeted at the intrinsic generation of thrombin.     

Factor XII auto-antibodies present in a patient with a B-cell lymphoma.

A 64-year-old woman was transferred for investigation of a mediastinal mass, biopsy of which showed a diffuse large B-cell lymphoma. She was also found to have an antiphospholipid antibody. The pre-operative coagulation screen showed a prolonged activated partial thromboplastin time, 71.3 s (normal range, 26-36 s), which was not corrected by the addition of normal plasma. The dilute Russell's viper venom time was positive. Anti-cardiolipin assay was strongly positive, immunoglobulin M was 153 AU; immunoglobulin G was normal, 3.1 AU. Assays of factors VIII, IX and XI showed higher concentrations with increasing dilutions in one-stage factor assays from 1: 10 to 1: 80 suggestive of an inhibitor. Factor XII was 9 U/dl and results were unaffected by increasing dilution, suggesting specific antibodies to factor XII. The factor XII antigen was 40 U/dl. The patient had immunoglobulin M auto-antibodies to factor XII.     

Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma

Recent investigations have suggested that the activation of factor IX by factor VII/tissue factor may be an important alternative route to the generation of factor Xa. Accordingly, we have compared the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and have studied the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H- labeled factor X to the plasma resulted, after a short lag, in burst- like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa, suggesting a feedback role for this enzyme, but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and (to a much smaller extent) factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. Variation of the factor IX or factor X concentrations permitted kinetic parameters for each activation to be derived. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin.     

Activation and inactivation of human factor X by proteases derived from Ficus carica

We investigated the effect of proteases derived from Ficus carica (common fig) on human blood coagulation. The milky sap (latex) of several Ficus (F.) species contain ficin, which is a mixture of proteases. Ficin derived from Ficus carica shortened the activated partial thromboplastin time and the prothrombin time of normal plasmas and plasmas deficient in coagulation factors, except plasma deficient in factor X (FX) and generated activated FX (FXa) in defibrinated plasma. Chromatographic separation of ficin from Ficus carica yielded six proteolytic fractions with a different specificity towards FX. We isolated two factor X activators with molecular masses of 23.2 and 23.5 kDa, and studied their action on purified human FX. Factor X was converted to activated FXbeta by consecutive proteolytic cleavage in the heavy chain between Leu178 and Asp179, Arg187 and Gly188, and Arg194and Ile195 (FX numbering system) with concomitant release of a carboxy-terminal peptide. The cleavage pattern of FXa degradation products in the light chain was influenced by Ca2+ and Mn2+. These data suggest the haemostatic potency of Ficus proteases is based on activation of human coagulation factor X.     

The activity state of factor VII in plasma: two pathways for the cold promoted activation of factor VII

The apparent amount of factor VII as determined in a one-stage test depends on the type of thromboplastin used: bovine thromboplastin only reacts with human factor VIIa whereas human thromboplastin interacts with unactivated human factor VII as well. Therefore the ratio factor VII activity as measured with bovine thromboplastin divided by the factor VII activity as assessed with human thromboplastin reflects the state of activation of factor VII in plasma. This approach was used to study the process of cold promoted factor VII activation and the involvement of different clotting factors therein. It could be shown that cold promoted activation does not occur in the absence of factors II and XII and is reduced for about 50% in factor IX deficient plasma. The other coagulation factors have a minor influence on the process. The results indicate that the cold promoted factor VII activation is the result of activation by both activated contact products and thrombin.     

Fletcher Factor Deficiency: A Report of Three Unrelated Cases

S ummary . The authors report three unrelated patients who apparently have a severe deficiency of the 'Fletcher factor'. None had a haemorrhagic tendency, and all had normal bleeding times, prothrombin times and thrombin times. All three showed defective intrinsic thromboplastin production, as evidenced by much prolonged kaolin activated partial thromboplastin time (PTT). None had deficiencies of factors VIII, IX, XI or XII. The prolonged PTT of each failed to shorten when his plasma was mixed with proven Fletcher deficient plasma. The plasma of one was assayed with plasma from a reference case of Fletcher deficiency and found to contain less than 1% of the normal activity. The prolonged PTT of each came down into or near the normal range on longer kaolin activation.     

Hemophilia as a defect of the tissue factor pathway of blood coagulation: effect of factors VIII and IX on factor X activation in a continuous-flow reactor.

The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a shear rate of 57, 300, or 1130 sec-1. Factor Xa in the effluent was determined by chromogenic assay. The flux of factor Xa (moles formed per unit surface area per unit time) was strongly dependent on wall shear rate, increasing about 3-fold as wall shear rate increased from 57 to 1130 sec-1. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2- to 3-fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor; however, when factors VIII and IX were present at their approximate plasma concentrations, factor Xa production rates were enhanced 15- to 20-fold. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.     

Consumption of Serum Factors and Prothrombin During Intravascular Clotting in Rabbits

We have studied the effect of experimental intravascular clotting upon Factors II, VII, IX, and X in rabbits. Animals were given sodium warfarin intravenously to block the synthesis of these factors and, 4 hours later, were infused with either dilute tissue thromboplastin or saline. The tissue thromboplastin induced intravascular clotting extensive enough to halve fibrinogen and platelet levels and to reduce markedly Factor V and Factor VIII levels. These changes resulted from a consumption during clotting of about 10 per cent of the available prothrombin. Each of the serum factors fell further during the infusion of tissue thromboplastin than during the infusion of saline; apparently, serum factors activated during clotting do not circulate in the rabbit. Net losses due to intravascular clotting equalled about 15 to 30 per cent of the Factors VII, IX, and X initially present. These data suggest that consumption during intravascular clotting may account for the low levels of Factors VII, IX, and X described in patients with diffuse intravascular clotting complicating septicaemia. The evidence of consumption of Factor IX raises the possibility that tissue thromboplastin may activate intrinsic clotting during hemostasis in vivo.     

Comparison of four commercially available activated partial thromboplastin time reagents using a semi-automated coagulometer.

Large numbers of activated partial thromboplastin time (aPTT) reagents are sold in the market. The phospholipid content and its source, nature and the amount of activators are highly varied in different aPTT reagents. The present study was undertaken to evaluate which of the four aPTT reagents commonly used is suitable as an all-purpose reagent for a modest haemostasis laboratory. Four aPTT reagents (reagent A, Platelin LS; reagent B, Silimat; reagent C, Actin FSL; reagent D, CK Prest) were tested against 75 different plasmas obtained from normal patients as well as from patients with different haemostatic problems. All the tests were conducted by one of us (S.S.) in duplicates. Different aPTT reagents missed different proportions of mild factor VIII and factor IX deficiency (36.4, 18.2, 4.6 and 13.6% for reagents A, B, C and D, respectively) and showed abnormal results with normal plasmas (i.e. more than 5 s prolongation) (29.2, 25, 8.3 and 12.5% for reagents A, B, C and D, respectively). All the reagents faithfully picked up moderate and severe factor VIII and factor IX deficiency. There was no difference among the four aPTT reagents regarding their ability to prolong aPTT to therapeutic dosage of heparin or in their ability to give comparable factor VIII or factor IX levels in one-stage aPTT-based assays. There were differences in aPTT reagents in their ability to pick up mild deficiency of coagulation factor VIII and factor IX. Some reagents showed abnormal aPTT results in mild cases of factor VIII and factor IX deficiency without producing a large number of falsely prolonged aPTT with normal plasma.