Residual Prothrombin Research Articles

Recombinant Factor VIIa (rFVIIa) Mediated Activation of Prothrombin Complex Concentrates (PCCs). Studies on the Comparison of Novoseven with a Biosimilar Product

IntroductionRecombinant factor VIIa (NovoSeven® Copenhagen, Denmark) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven® is also developed for same indication (AryoSeven™, AryoGen, Tehran-Iran). The two products exhibit comparable compositional and biochemical profile. To compare the activation profile of NovoSeven® and AryoSeven™, two commercially available protein complex concentrates (PCCs) were used. The activation profiles were studied utilizing SDS- gel electrophoresis profile and immunoblotting using anti-rabbit recombinant thrombin antibody.Materials & MethodsNovoSeven®, AryoSeven™, Profilnine® SD (Grifols Biologicals Inc. USA), and Beriplex® (CSL Behring Canada, Inc.) were obtained commercially. Recombinant human thrombin antibodies were generated in rabbits. Recothrom® was commercially obtained (Zymogenetic, Seatle,Wa- USA and Medicine Company ,Parsippany, NJ –USA). Tissue factor RecombiPlasTin 2G was obtained from Instrumentation Laboratory Company (Bedford- USA).NovoSeven® and AryoSeven™ were diluted with saline to make working solution of 1mg/ml. Further dilutions were made with saline to obtain 0.5 µg/ml, 1.25µg/ml, and 2.5µg/ml rFVIIa preparations. To make working solution of PCCs, PCCs were diluted to 10 U/ml in saline. The sample mixtures contain 2U/ml PCCs, 0.1, 0.25, 0.5 µg/ml rFVIIa, and with RecombiPlasTin 2G at 1:5 ratio. The samples mixtures incubated in 5, 15, and 30 minutes at 37°c. SDS-PAGE (Pierce Biotechnology) and immunoblotting carried against Recothrom®.ResultsProfilnine® SD activated by RecombiPlasTin 2G resulted in conversions of prothrombin to prethrombin and thrombin at 5 to 30 minutes incubation time as evident by the generation of distinct bands at 50kDa and 36 kDa. However, addition of rFVIIa at final concentration range of 0.25 and 0.5µg/ml to the same mixture resulted in total conversion of prothrombin to thrombin at 30 minutes with a doublet bands at 36 kDa. Recombinant FVIIa alone failed to generate thrombin in native Profilnine® SD at 5 to 30 minutes as evident by the absence of 36 kDa band in the SDS-PAGE and Immunoblotting studies. Further studies comparing NovoSeven® and AryoSeven™ (FC: 0.1µg/ml) in the activated Profilnine® SD resulted in a comparable and progressive generation of thrombin at 5 and 15 minutes incubation time. At 5 and 15 minutes residual prethrombin bands were noted which was less intense at 15 minute incubation time in both rFVIIa preparations. Although rFVIIa alone unable to generate thrombin in native Beriplex®, the presence of rFVIIa in activated Beriplex did not change the activation profile. Activated Beriplex® by RecombiPlasTin 2G resulted in complete conversion of prothrombin to thrombin at 30 minutes incubation time. A complete generation of thrombin was noted in both activated PCCs in the presence of rFVIIa at final concentrations of 0.25µg/ml and 0.5µg/ml. At 5 minutes residual prothrombin and prethrombin bands were noted in the presence of rFVIIa at final concentration of 0.1µg/ml. However, at 15 minutes prothrombin bands were completely disappeared and prethrombin bands were less intense at same concentration of 0.1µg/ml of rFVIIa. The results obtained by Western blot analysis confirmed the differential activation of Profilnine® SD and Beriplex®. However, both the NovoSeven® and AryoSeven™ exhibited similar activation profiles.ConclusionsThese results indicate that the activation of PCCs by both AryoSeven™ and NovoSeven® result in comparable generation of thrombin. Furthermore, while both rFVIIa produced similar activation profile, PCCs based differences in the activation profile were noted. These studies provide an in vitro profiling of the effects of activated FVIIa on the generation of thrombin and related enzymes to demonstrate biosimilarity of the branded and generic versions of these haemostatic products. DisclosuresNo relevant conflicts of interest to declare.

Some hemostasis variables at the end of the population distributions are risk factors for severe postpartum hemorrhages.

Severe postpartum hemorrhages (PPH) represent a significant cause of maternal morbidity/mortality, but little is known about its hemostasis-related risk factors. Among the 32 463 women enrolled in the NOHA First cohort, 317 developed severe PPH (S-PPH group), 1269 non-severe PPH (NS-PPH group) and the remaining individuals were considered as control women (C group). We performed a case-control study, including 317 triplets of women allocated from the three groups that shared the same clinical characteristics as the S-PPH group. From values obtained 6-9 months after delivery, low (but not-deficient) levels of fibrinogen, von Willebrand factor (VWF) antigen, factor (F) XI, platelet CD42b, TRAP-induced increase of platelet CD41a and high values of serum residual prothrombin activity or closure aperture times using the collagen-ADP cartridge on the PFA-100 system, and blood group O, were independently associated with a significant risk of severe PPH. Being positive for at least two of these eight variables was found in 1.6%, 3.5% and 20.8% of the women from the C, the NS-PPH and the S-PPH groups, respectively, the odds ratio for S-PPH in such a case being 16.4, 95%CI (6.5-41), P < 0.0001. Women with some hemostasis-related variables at the low or high end of the population distributions are prone to the severe forms of PPH. Clinical trials will allow us to know if acting on these risk factors can lower the clinical severity of PPH.

Scott syndrome: an inherited defect of the procoagulant activity of platelets

Anionic phospholipids, chiefly phosphatidylserine, are essential for the assembly of the characteristic enzyme complexes of the blood coagulation cascade at the surface of stimulated platelets and derived microparticles. In the resting cell, these phospholipids are sequestered in the inner leaflet of the plasma membrane. Scott syndrome is an extremely rare bleeding disorder that confirms the essential role of these anionic procoagulant phospholipids. In Scott patients, phosphatidylserine externalization and microparticle shedding are dramatically impaired. This functional deficiency is clearly evidenced by the measurement of residual prothrombin in serum. The recent detection of a familial Scott syndrome testifies to the genetic origin of the defect. Symptomatic Scott patients present provoked hemorrhages and are probably homozygous for the disorder whereas asymptomatic children are probably heterozygous. The Scott phenotype can be detected in platelets, red cells and lymphocytes by functional prothrombinase assay and flow cytometry. Intermediate degrees of phosphatidylserine exposure and vesiculation are observed in cells from the asymptomatic heterozygous offspring when compared to those from their homozygous defective parent and healthy subjects. The functional and molecular characterization of mutated element(s) in Scott syndrome should be of valuable help for the understanding of phospholipid transmembrane migration, also termed flip-flop, its possible links with membrane vesiculation, and the eventual implications in thrombotic or apoptotic processes.

Mechanisms for the antithrombotic activity in man of low molecular weight heparins (LMWHs).

Although LMWH have been widely used for several years, the mechanisms of their anti-thrombotic action are not well understood. This is due in part to the misconception that in vitro coagulation results can be extrapolated to the in vivo antithrombotic condition. Thus, the mode of action of heparins on thrombin generation in plasma after addition of increasing amounts of unfractionated heparin (UH) or LMWH has been investigated in several studies, but a distinction has to be made between the effects on the intrinsic and the extrinsic system. The same applies for experiments conducted with platelet-poor plasma (PPP) and platelet-rich plasma (PRP). These experiments show that inhibition of prothrombin activation in PPP is more pronounced in the intrinsic system than in the extrinsic system for both types of heparin. This inhibition has been attributed mainly to their antithrombin activity which reduces factor V and factor VIII activation and its related positive feedback on thrombin formation. The comparison of the effects of low doses of LMWH and UH in citrated PRP indicate that LMWH are more active than UH because they are more resistant to platelet neutralization by platelet factor 4. Moreover, in vitro study will neglect the role of pharmacokinetic parameters which are important determinants of the antithrombotic activity of heparins. Another important difference between in vitro and in vivo studies lies in the fact that sc injection of both heparins will release tissue factor pathway inhibitor (TFPI) in the blood. We have used native whole blood for in vitro and ex vivo experiments. This offers the advantage of studying heparin activity in the presence of platelets and calcium. In this condition the inhibition of pro-thrombin and factor VII activation during blood coagulation in a glass tube (intrinsic system) was judged by measuring residual prothrombin and factor VIIa in serum obtained 2 to 4 hours after clotting. At prophylactic doses, LMWHs in contrast to UH significantly inhibit prothrombin activation. Although thrombin inhibition seems essential for the antithrombotic activity of both heparins, reduction of thrombosis is a global effect to which "both anti-IIa and anti-Xa activity contribute but to a different exent" (C. Hemker et al.). It should be noted that the clearance half-life of anti-Xa activity is significantly longer than that of anti-IIa. The efficacy of a single daily injection of LMWH in the prophylaxis of thrombosis is not logical in light of the short half-life of anti-IIa activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Acquired Hypoprothrombinemia Due to Nonneutralizing Antibodies to Prothrombin: Mechanism and Management

A patient developed bleeding due to an acute acquired specific prothrombin deficiency. Unlike previously described patients, this patient had no evidence of an associated lupus anticoagulant. Prothrombin activity and antigen were decreased concordantly and the patient's plasma did not neutralize the activity of added prothrombin or interfere with its measurement by electroimmunoassay. Nevertheless, immunoelectrophoresis and experiments using 125I-prothrombin revealed a prothrombin-binding antibody. The residual prothrombin in the patient's plasma was in the form of a prothrombin-antibody complex. Administration of adrenal corticosteroids was associated with a rise in prothrombin activity and cessation of bleeding, but circulating prothrombin was still bound to the antibody. This suggests that non-neutralizing antibodies to prothrombin cause plasma prothrombin deficiency because of a rapid clearance of prothrombin-antibody complexes, which is slowed by adrenal corticosteroids. The antibody had a relatively low affinity for prothrombin (Kd 5 to 8 X 10(-7)) and was transient. It is possible, therefore, that the antibody arose not to prothrombin itself, but to an antigen sharing an epitope with prothrombin.

Effect of oral contraceptive use on platelet prothrombin converting (platelet factor 3) activity

Platelets obtained from women using estrogen-progesterone oral contraceptives are evaluated for activity as cofactor for Factor Xa-catalyzed prothrombin activation. Donor platelet-rich plasma is diluted into a standard platelet-poor plasma to minimize variations in plasma Factor X and prothrombin concentrations. Activation is initiated with Russell's viper venom, arrested at intervals with EDTA, and residual prothrombin is measured. Compared with controls, platelets from women using oral contraceptives promote an increase in both rate and extent of prothrombin activation, with control and test groups falling into non-overlapping populations. The increased prothrombin activation correlates with an increase in antithrombin III consumption. Enhanced antithrombin III consumption explains the decreased serum antithrombin III levels among women using oral contraceptives.

Evidence that washed human platelets possess factor-X activator activity.

Summary. Human platelets, washed by repeated albumin density gradient centrifugation or by gel filtration, aggregate upon addition of normal human serum and calcium chloride, due to generation of thrombin from the small amount of residual prothrombin present in normal serum. The rate of thrombin formation is dependent on the factor V provided by the platelets as shown by a marked decrease in formation when platelets from a subject with congenital factor‐V deficiency are used instead of normal platelets. When phospholipid, factor V (bovine serum absorbed with barium sulphate), calcium chloride and fibrinogen were added to serum, no thrombin formation was observed, indicating that serum itself was devoid of factor Xa. In addition, when washed platelets and calcium chloride were mixed with a factor‐X deficient serum or with a highly purified prothrombin preparation devoid of factor X, hardly any thrombin generation occurred, indicating that the washed platelets did not supply factor Xa. Therefore activated factor X is probably generated in the mixture containing washed platelets, normal serum and calcium chloride. Activation of factor X via the intrinsic pathway is unlikely since (a) washed platelets and normal serum are devoid of factor VIII, and (b) washed platelets and/or serum obtained from subjects with factor‐VIII or factor‐IX deficiency or with factor‐VIII antibodies behaved in the same way as normal platelets or serum in the test system. Activation of factor X via the extrinsic pathway is also unlikely since (a) serum and platelets from a congenitally factor‐VII deficient subject behaved normally in the test system, and (b) rapid thrombin generation occurred when serum was replaced by purified prothrombin and purified factor X devoid of factor VII. These findings indicate that washed human platelets possess factor‐X activator activity. Platelet factor 3 is not strongly implicated in the generation of thrombin in this test system since prostaglandin El, which reduces its availability, had no influence on the reaction.

Quantitative Determination of Platelet Factor 3 Activity

SummaryA method for the quantitative determination of platelet factor 3 (PF 3) is described. It is based on the prothrombin consumption and allows the determination of the total PF 3, of the PF 3 made available under various conditions and of PF 3 like activity.The material to be tested is recalcified in the presence of a normal PPP pool to maintain the intrinsic clotting factors in the optimal concentration range. After an appropriate incubation the coagulation is stopped by a citrate solution and the clot removed. The residual prothrombin activity in citrated serum is indirectly proportional to the PF 3 activity. This activity can be expressed in per cents by means of a reference curve which can be established with Cephaloplastin (Dade) dilutions. The 100% value is given by the total average PF 3 activity when the platelet count in PRP is adjusted to 100,000 platelets/mm3. An appropriate correction is needed when the prothrombin concentration is too low in the tested PRP.In 50 healthy blood donors the PF 3 activity ranged between 70-130% with quite small day to day variations.

Determination of platelet factor-3 by a modified prothrombin consumption test.

A method for the determination of platelet factor‐3 by means of a prothrombin consumption test has been developed. The suspension of platelets with a concentration of 50–100,000/μl is washed three times. The results are expressed in percentage of residual prothrombin. The method is also useful in thrombocytopenic states.

Some Effects of Purified Autoprothrombin C in Blood Clotting

SummaryPurified autoprothrombin C was added to rabbit blood or recalcified plasma in order to measure the exact influence on clotting time. At a concentration of 10 u of autoprothrombin C per ml blood there was clotting in 13 sec. At that concentration recalcified plasma clotted in 7 to 8 sec. In the acceleration of the whole blood clotting time with autoprothrombin C, the effect was greater in glass tubes than in silicone coated tubes. The difference was probably due to lipids from platelets. With autoprothrombin C at a concentration of 10 u/ml reaction mixture, the prothrombin time and partial thromboplastin time of plasma was brought down to 3 to 4 sec. The partial thromboplastin time supplied the most sensitive conditions for detecting autoprothrombin C activity. Less than 2 × 10−5 micrograms of autoprothrombin C were detected. Rabbits that were treated with Coumadin had their prothrombin concentration reduced to 9 u/ml plasma, as measured by two-stage analysis. Such plasma yielded twice as much thrombin when purified autoprothrombin C and purified Ac-globulin were used in the assay to determine the thrombin potential. Even when the prothrombin concentration was brought to low levels with Coumadin a partial thromboplastin time or prothrombin time of 7 sec was easily obtained by using autoprothrombin C as procoagulant. The important effect of Coumadin or related drugs is the reduction of prothrombin concentration. This involves a lowering of the autoprothrombin C potential as well as the thrombin potential of plasma, and also the amount of inhibitor that can be obtained from prothrombin.Normal blood was found to contain prethrombin in small amounts as well as prothrombin. The synthesis of prothrombin was stopped with Coumadin and it is likely that the residual prothrombin was in part utilized by degradation to prethrombin, inhibitor and autoprothrombin III. At the height of the anticoagulant effect the prethrombin concentration was higher than the prothrombin concentration. With the resumption of prothrombin synthesis, when vitamin K was given, it may be that prethrombin, inhibitor, and autoprothrombin III was first produced in the liver and some of the latter entered the blood stream to compensate for substrate deficiency. Free autoprothrombin III would account for the short prothrombin time observed before prothrombin concentration rose.

Addition of Prothrombin to Blood from Dogs Receiving Dicumarol

SummaryThe prothrombin concentration of dog plasma was lowered by giving large doses of Dicumarol. The dog blood was then mixed with progressive increments of purified bovine prothrombin. When the concentration of prothrombin was equivalent to normal the whole blood clotting time and the prothrombin time were normal. The purified prothrombin supplied all that was essential and did not add detectable amounts of extraneous pro coagulant power. The residual prothrombin in the serum was generally higher when clotting occurred in silicone lined test tubes than in glass. In silicone tubes very little hemophilia B (factor IX, autoprothrombin II) activity developed. This activity was found in the glass tubes in a concentration proportional to the original prothrombin added. The transformation of prothrombin to autoprothrombin II occurred in plasma when there was a glass surface.

Blood clotting defect in hibernating ground squirrels (Citellus tridecemlineatus).

In investigating the hypocoagulability of the blood during hibernation, multiple plasma-clotting factors, numbers of platelets, and hematocrits were determined in 17 hibernating 13-lined ground squirrels and compared with levels in 23 nonhibernating ground squirrels. Human plasma was used as the standard in procoagulant assays. At the time of withdrawal of the blood from the aortas of hibernating animals, their rectal temperatures ranged from 6.9 to 8.6 C. Significant differences between active and hibernating animals were found in the whole-blood clotting times (210 and 315 sec), partial thromboplastin times (45.5 and 109.3 sec), plasma prothrombin (443 and 698 U/ml), 1-hr serum residual prothrombin (27 and 449 U/ml), factor VIII (165 and 35%), factor IX (359 and 188%), and blood platelet concentration (445,150 and 47,940/mm3). No decisive changes were demonstrated in the prothrombin times or levels of factors V, VII, X, XI, XII, or fibrinogen. Inhibitors were not detected. Both thrombocytopenia and plasma defects seemed to contribute to the impairment of the blood-clotting mechanism during hibernation.

The Prothrombin Consumption Time Test in Thrombocytopenia

SummaryThe serum from adult platelet-poor plasma has a residual prothrombin content nearly as high as that of the plasma when measured by either the prothrombin consumption test or the two-stage method of Ware and Seegers. Good agreement between the two methods is obtained when the prothrombin concentration is altered by such means as absorption with Ca3(PO4)2. The prothrombin consumption test is a simple but delicate measure of the function of platelets in the generation of intrinsic thromboplastin.

SURGERY IN A HEMOPHILIAC CHILD WITH INTRACRANIAL HEMORRHAGE

THE UNUSUAL bleeding tendency in hemophiliacs has been known since biblical times, and its hazards have been recognized in even such simple surgical procedures as circumcision.1 Perhaps the most dangerous complication of hemophilia is bleeding into the central nervous system.2 It therefore seems worthwhile to report the case of a 2-year-old hemophiliac who survived several intracranial hemorrhages, with two surgical interventions, but who 4 months later had a fourth and fatal hemorrhage. CASE REPORT History D. H. was a 2-year-old hemophiliac with numerous admissions to the Kaiser Foundation Hospital, Los Angeles, for bleeding episodes. He was born at another hospital, was circumcised shortly after birth and had excessive bleeding following this. His first admission was at the age of 1 year for bleeding following a tongue bite. At that time he had an abnormal result of a prothrombin consumption test, with 55% residual prothrombin in the serum. The prothrombin consumption was corrected by fresh normal plasma and barium sulfate adsorbed plasma, but not by serum or plasma from a known hemophiliac type A. The diagnosis of hemophilia type A was thus established. He had two brothers who were investigated and found to be normal. There was no abnormal bleeding tendency on his father's side, but two uncles of his mother and two first cousins were said to be "bleeders." No further details were known. Physical and Laboratory Findings The boy was admitted to the hospital on the evening of October 13, 1957, with a history of vomiting and progressive lethargy of 2 days' duration.

Effects of Ethylenediaminetetraacetic Acid (EDTA) on Prothrombin Consumption

Whole human blood was treated with varying concentrations of ethylenediaminetetraacetic acid to determine the effect upon prothrombin consumption. The prothrombin consumption test was combined with the Dreskin two-stage prothrombin test to determine residual prothrombin as a function of incubation time. Ethylenediaminetetraacetic acid in high concentrations inhibited prothrombin consumption; in one concentration prothrombin consumption was accelerated, and in the lower concentrations no effect was observed. These data indicate that EDTA has a diphasic action upon the utilization of prothrombin as a part of its diphasic action in whole blood coagulation.

Coagulation properties of canine thoracic-duct lymph.

Coagulation studies were made on thoracic-duct lymph obtained from semifasting healthy adult dogs. Prothrombin, fibrinogen, antihemophilic factor (AHF) and plasma thromboplastin component (PTC) were found to be present in lymph in concentration of about 30–60% of plasma levels. One-stage type clotting tests using either complete or partial thromboplastins give variable results. Some samples of lymph are as active in these tests as is plasma while some are considerably less active. The lymph samples are not fully active at the time of collection. On exposure to glass surfaces in the presence of anticoagulant, the clotting time becomes shorter during the first 20–40 minutes. Coagulating lymph has a high residual prothrombin even after 18–24 hours in glass containers. Thoracic-duct lymph contains sufficient thromboplastic materials so that adequate amounts of thrombin can form to produce a fibrin clot, but it does not contain the thromboplastic materials required for complete prothrombin utilization. The study of lymph coagulation appears to be an important aspect of the general process of coagulation since thrombocytes appear to play a minor role in its coagulation properties.

Rivanol, resin and the isolation of thrombins.

HUMAN, cow, and horse thrombins were quickly fractionated from citrate- or bio-activated prothrombin preparations by precipitation of residual prothrombin and inert protein(s) with the cationic dye 6,9-diamino-2-ethoxyacridine lactate (often referred to as rivanol, available under the trade name ‘Ethodin’ from the Winthrop Laboratories, New York, New York). This is a general procedure previously applied in purifying γ-globulin1, β1-metal-combining globulin2, and caeruloplasmin3. A more efficient yet rapid fractionation, however, was achieved by the adsorption of preparations containing thrombin on filter cakes or short columns of ‘IRC-50’ (‘XE-64-Rivanol’), this resin form being prepared by stirring ‘XE-64-Na+’ with an excess of rivanol. After the impurities, including other rivanol-soluble proteins, were washed off, the thrombins were eluted with 0.15 M calcium chloride. The thrombins were recovered from all eluates by acetone precipitation.

Viscous metamorphosis, clot retraction and other morphologic alterations of blood platelets

Some samples of washed platelets undergo viscous metamorphosis (VM) in oxalated or citrated plasma, oxalated serum or serum poor in prothrombin. Other samples only undergo VM when serum containing residual prothrombin is used and thrombin is evolved when platelets are added. Thrombin produces VM at pH 7.4 but not at pH 6.1 or in isotonic saline. Treatment of thrombin with diisopropylfluorophosphate (DFP) or addition of heparin plus serum inhibits VM. Spreading of platelets upon glass, and balloon formation are not closely correlated with VM. Clot retraction in a purified system often occurs without added buffer or glucose, but is always very marked in the presence of both buffer and glucose. Calcium binding anticoagulants prevent VM and clot retraction. The inhibition is reversed by calcium and, to a lesser extent, magnesium. Cyanide (4 or 5 x 10-3 m) does not inhibit VM or retraction in a purified system but inhibits retraction of platelet-rich plasma, especially when it is clotted with thrombin. At this concentration, fluoride has a variable effect. Mercuric chloride is an effective inhibitor of both VM and retraction at concentrations of 10-4 m and below. Submitted on November 20, 1958

The Comparative Effect of Platelets on Prothrombin Utilization from Dogs in the Degenerative and Regenerative Phase of Irradiation Bone Marrow Aplasia

Under specific conditions, a quantitative relationship between platelet concentration and amount of prothrombin utilized in the coagulation of shed blood in glass tubes was demonstrated by Buckwalter et al. (1). In their studies, canine and human blood was collected into cold, siliconed glassware without the aid of anticoagulants. Platelet-rich plasma was obtained by slow centrifugation in the cold, and the platelet count was adjusted by differential centrifugation and dilution with plateletpoor plasma. The coagulation was initiated by transfer of platelet plasma mixture to chemically clean Pyrex glassware at room temperature, coagulation stopped at intervals by the addition of sodium citrate, and the residual prothrombin assayed by a two-stage prothrombin determination (2). This procedure was modified and applied by Jackson et al. (3) in the investigation of the relation of the platelet count to prothrombin utilization and the hemorrhagic tendency in dogs exposed to lethal whole-body irradiation. In these studies, it was not necessary to resort to differential centrifugation because the platelet count progressively decreases to almost zero by the tenth day as a result of irradiation aplasia of the marrow. In general, there was a parallelism in the decrease in the platelet count and the diminished utilization of prothrombin. However, it appeared that, under these conditions, the utilization of prothrombin diminished more rapidly than the platelet count. In further studies, Cronkite et al. (4) confirmed these observations and found that in the prothrombin utilization returned much more rapidly to normal than did the platelet count. In addition, Ferguson et al. (5), using injected radiogold, has reported that prothrombin utilization is depressed before there is a significant decrease in the platelet count. The above observations can be interpreted as indicating that platelets are less active than normal in accelerating prothrombin utilization in the degenerative phase and more active than normal in the regenerative phase. An alternative explanation in the degenerative phase would be the presence of abnormal anticoagulant or increased amounts of normally occurring lipid antithromboplastins.

Plasma thromboplastin component (PTC) potency of plasma fractions.

Conclusions1. The PTC factor is concentrated in fractions III and IV-1 of the plasma proteins, Small quantities are found in fractions I and IV-4. None is found in fractions II and V. 2. Since fraction III contains thrombin and fraction IV-1 is poorly soluble, neither is suitable for intravenous administration. 3. The intravenous administration of fraction IV produced marked shortening of the whole blood coagulation time and reduction in residual serum prothrombin in PTC deficiency. The results appear superior to those which can be achieved by the intravenous administration of fraction I in hemophilia.