Hemophilia A: polymorphism detectable by a factor 8 antibody.

Plasma from 54 patients with hemophilia A was tested for neutralizing activity with a human antibody to factor VIII. The plasma from 52 patients had no demonstrable neutralizing activity. Two plasma samples had neutralizing activity equivalent to that of normal plasma despite the lack of factor VIII clotting activity. Apparently, most patients with hemophilia A do not synthesize factor VIII, whereas a few synthesize an inactive molecule with a presumed genetic structural mutation of the active site but with antigenic determinants in common with normal factor VIII. Thus, hemophilia A is a disease caused by more than a single genetic mechanism.

Mechanism of the anticoagulant effect of warfarin as evaluated in rabbits by selective depression of individual procoagulant vitamin K-dependent clotting factors.

We have evaluated the contribution of depression of individual procoagulant vitamin K-dependent clotting factors to the ability of warfarin to protect rabbits against tissue factor-induced coagulation. Mean activities of individual procoagulant factors were determined, in assays with rabbit substrates, for a group of rabbits achieving a protective degree of anticoagulation with warfarin. Values were: factor VII, 12%; factor IX, 7%; factor X, 14%, and prothrombin, 13%. The effect upon tissue factor-induced coagulation of selective immunodepletion of each factor to a comparable level was then evaluated. Immunodepletion of plasma factor X or prothrombin, but not of factor VII or factor IX, protected otherwise normal rabbits against tissue factor-induced coagulation. Next, we determined the effect upon the protection in warfarin-treated rabbits of selectively restoring factor X or prothrombin before infusing tissue factor. When either factor was selectively restored, warfarin's protective effect was abolished. Moreover, selective restoration of prothrombin sensitized warfarin-treated rabbits to coagulation more severe than observed in nontreated control rabbits. One may extrapolate from these data that depression of both factor X and prothrombin are required for warfarin's clinical antithrombotic efficacy and that depression of plasma prothrombin is particularly important.

Factor V anticoagulants: clinical, biochemical, and immunological observations.

A patient who had received multiple transfusions for complications of acute hemorrhagic pancreatitis developed a potent factor V anticoagulant with bleeding due to defective hemostasis. Despite its potency, the anticoagulant disappeared within 15 days of its first manifestation. A second patient with adenocarcinoma of the colon developed an anticoagulant to factor V postoperatively after a single blood transfusion. The anticoagulants appeared to react stoichiometrically with factor V in normal plasma in vitro. They had the physicochemical properties of immunoglobulins, and their activity was neutralized by antihuman immunoglobulin antiserum. One anticoagulant appeared to be slightly more active against homologous than against autologous factor V, but it also inhibited heterologous factor V. Both anticoagulants progressively inactivated intrinsic prothrombin activator formed from normal reagents in the incubation mixture of the thromboplastin generation test, thus confirming that factor V is required for the effective action of the intrinsic prothrombin activator. Since the anticoagulants were immunoglobulins whose activity was consumed in their reaction with factor V, consumption of anticoagulant activity was used to detect factor V antigenic material in test materials. Human serum without factor V clotting activity was found to consume anticoagulant activity, i.e., to contain inactive factor V antigenic material. Plasma from two patients with hereditary factor V deficiency (parahemophilia) failed to consume significant anticoagulant activity. Thus, the lack of factor V activity in these patients represents a deficiency of factor V molecules rather than the synthesis of a defective molecule with impaired clotting activity.

Activation of human factor VII in plasma and in purified systems: roles of activated factor IX, kallikrein, and activated factor XII.

Factor VII can be activated, to a molecule giving shorter clotting times with tissue factor, by incubating plasma with kaolin or by clotting plasma. The mechanisms of activation differ. With kaolin, activated Factor XII (XII(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-deficient plasma, was partially activated in prekallikrein and high-molecular weight kininogen (HMW kininogen)-deficient plasmas, but was activated in other deficient plasmas. After clotting, activated Factor IX (IX(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-,HMW kininogen-, XI-, and IX-deficient plasmas, but was activated in Factor VIII-, X-, and V-deficient plasmas. In further studies, purified small-fragment Factor XII(a) (beta-XII(a)), kallikrein, and Factor IX(a) were added to partially purified Factor VII and to plasma. High concentrations of beta-XII(a) activated Factor VII in a purified system; much lower concentrations of beta-XII(a) activated Factor VII in normal plasma but not in prekallikrein or HWM kininogen-deficient plasmas. Kallikrein alone failed to activate partially purified Factor VII but did so when purified Factor IX was added. Kallikrein also activated Factor VII in normal, Factor XII-, and Factor IX-deficient plasmas. Purified Factor IX(a) activated partially purified Factor VII and had no additional indirect activating effect in the presence of plasma. These results demonstrate that both Factor XII(a) and Factor IX(a) directly activate human Factor VII, whereas kallikrein, through generation of Factor XII(a) and Factor IX(a), functions as an indirect activator of Factor VII.

Osmotic blood-brain barrier disruption. Computerized tomographic monitoring of chemotherapeutic agent delivery.

The present study describes a canine model of transient reversible blood-brain barrier disruption with hyperosmolar mannitol infusion into the internal carotid artery. Studies in this model show that osmotic blood-brain barrier disruption before intracarotid infusion of methotrexate results in markedly elevated (therapeutic) levels of drug in the ipsilateral cerebral hemisphere. Levels in the cerebrospinal fluid correlate poorly and inconsistently with brain levels. Computerized tomograms in this canine model provide a noninvasive monitor of the degree, time-course, and localization of osmotic blood-brain barrier disruption.

Fibrinogen response to turpentine and endotoxin in busulfan-treated rabbbits.

The response of the plasma fibrinogen level to the subucutaneous injection of turpentine and to the intravenous injection of endotoxin was measured in normal rabbits and in rabbits made granulocytopenic and thrombocytopenic with busulfan. Plasma fibrinogen levels rose sharply in both normal and busulfan-treated rabbits and the extent of the rise in fibrinogen level after turpentine. As discussed herein, these data are consistent with the hypothesis that material from granulocytes plays a pathophysiologic role in the stimulation of fibrinogen synthesis in inflammation and after tissue trauma.

Studies of the factor Xa-dependent inhibitor of factor VIIa/tissue factor (extrinsic pathway inhibitor) from cell supernates of cultured human umbilical vein endothelial cells.

Cultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VIIa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

Antibody-induced acute factor X deficiency: clinical manifestations and properties of the antibody.

A patient is described with serious bleeding due to a transient selective deficiency of factor X. Crossed immunoelectrophoresis of patient's plasma with anti-factor X antibody revealed an abnormal factor X arc suggestive of the presence of plasma factor X/anti-factor X immune complexes. A similar abnormal arc was obtained on adding the patient's IgG to normal plasma. Immunoblotting of factor X after reduced SDS-PAGE revealed that the patient's IgG bound to the light chain of intact factor X but not Gla-domainless factor X. The patient's IgG inhibited activation of factor X by VIIa/tissue factor (TF), by IXa/VIIIa/phospholipid complex, and by Russell's viper venom. The IgG failed to inhibit the proteolytic activity of factor Xa towards a chromogenic substrate. However, under reaction conditions of limited factor Xa availability, the IgG could be shown to impair hemostatic functions of factor Xa that require the participation of its light chain: activation of prothrombin by prothrombinase; activation of factor VII bound to TF; and inhibition of VIIa/TF activity by factor Xa/tissue factor pathway inhibitor complexes. A few earlier patients have been described with transient, selective factor X deficiency and serious bleeding, but in only one was evidence obtained of an antibody against factor X. It will be of interest to learn whether use of the techniques described in this report will permit the identification of immunoglobulin with similar binding and functional properties in future patients with this rare syndrome.

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide.

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

Functional properties of factor VIIa/tissue factor formed with purified tissue factor and with tissue factor expressed on cultured endothelial cells.

Factor VIIa (F. VIIa)/tissue factor (TF) function was examined using purified human TF reconstituted into mixed phospholipid vesicles and TF expressed on cultured human umbilical vein endothelial cells (HUVEC) treated with thrombin. In reaction mixtures containing either type of TF, F. VIIa, 10 nM, either 3H-factor X or 3H-factor IX, 88 nM, and Ca2+, 5 mM, F. VIIa/TF activated factor X (F. X) several fold faster than it activated factor IX (F. IX). Adding heparin, 1 U/ml, increased rates of activation of both substrates and F. X remained the preferred substrate. Adding plasma at concentrations of 5% or above inhibited factor VIIa/TF catalytic activity. Inhibition was shown to require F. Xa as a cofactor, was prevented by antibodies to extrinsic pathway inhibitor (EPI), and was reversible by decalcification. Thus, with factor VIIa/TF formed with both types of TF, EPI appeared responsible for inhibition induced by plasma. Our data indicate that functional properties of factor VIIa/TF as delineated in reaction mixtures made with purified TF reconstituted into mixed phospholipid vesicles also hold for factor VIIa/TF activity on the surface of cultured HUVEC.

Differences in the interactions of lupus anticoagulant IgG with human prothrombin and bovine prothrombin.

Lupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin that the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell's viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing > 50% of inhibition. In contrast, only one LA IgG markedly inhibited (> 50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet aggregate ratios--standardization of technique and test results in patients with myocardial ischemia and patients with cerebrovascular disease.

The technique for measuring platelet aggregate ratios described by Wu and Hoak (1974) was evaluated in normal subjects. The following had no influence upon the test result: age, sex, fasting versu the postprandial state, and the degree of stasis prior to drawing the sample. Variance within subjects was small compared to variance between subjects (0.009 versus 0.0053, p less than 0.01). Platelet aggregate ratios were then measured in 36 patients with coronary artery disease hospitalized with acute chest pain. Their mean platelet aggregate ratio of 0.86 was identical to the mean ratio for 47 normal subjects. Greater variability was found within patients (between samples) than within the normal subjects. This observation raises doubts about the significance of a single measurement of platelet aggregate ratio in such acutely ill patients. Mean platelet aggregate ratios measured daily did not differ over a 7-day period between 11 patients who developed a myocardial infarction and 10 patients who did not. A normal mean platelet ratio was also found on a single measurement from 30 patients with a history of completed stroke (0.87) and from 11 patients with a history of transient ischemic attacks (0.92).

Effects of endotoxin in cortisone-treated rabbits with a hereditary deficiency of the sixth component of complement (C6 deficiency).

Endotoxin was infused into normal rabbits and C6 deficient rabbits prepared with cortisone for the generalized Shwartzman reaction. Endotoxin produced profound granulocytopenia and moderate thrombocytopenia in both normal and C6 deficient rabbits. In normal rabbits endotoxin consistently produced extensive intravascular clotting. In C6 deficient animals endotoxin resulted in intravascular clotting of variable extent. In one group of eight C6 deficient rabbits mean fibrinogen levels fell 0.67 g per 1 over 6 hrs after endotoxin and four of eight animals developed a generalized Shwartzman reaction. In a second group of seven C6 deficient rabbits mean fibrinogen level fell only 0.17 g per 1 over 6 hrs and one animal developed a generalized Shwartzman reaction. Values for mean fibrinogen consumption, calculated from plasma fibrinogen levels and rate of disappearance of 25I-fibrinogen, were as follows: normal animals infused with saline, 10 mg per kg; C6 deficient animals infused with endotoxin, 58 mg per kg. Fibrinogen consumption after endotoxin was found to be related to granulocyte levels prior to endotoxin, which determined the number of granulocytes disappearing from the blood after endotoxin. The data indicate that C6 deficiency in the rabbit does not prevent intravascular clotting and the generalized Shwartzman reaction.

Heparin-releasable and platelet pools of tissue factor pathway inhibitor in rabbits.

Earlier studies from this laboratory have established that tissue factor pathway inhibitor (TFPI) functions as a natural anticoagulant protecting rabbits from intravascular coagulation triggered by the exposure of blood to small amounts of tissue factor. In addition to the TFPI circulating in plasma, humans have been shown to have heparin-releasable and platelet pools of TFPI. In order better to extrapolate from studies carried out in rabbits to an understanding of human hemostasis, we have examined the presence and extent of heparin-releasable and platelet pools of TFPI in rabbits. We find that in the rabbit the heparin-releasable pool of TFPI activity, as measured in a capacity assay, may be smaller relative to the plasma pool than in humans; that the platelet pool of TFPI activity is comparable to that of humans; and that rabbit TFPI, unlike human TFPI, has the same apparent molecular mass in all vascular pools. These studies extend our understanding of the properties of TFPI in rabbits and the appropriateness of using the rabbit for studies of TFPI relevant to human hemostasis.

Modifications of extrinsic pathway inhibitor (EPI) and factor Xa that affect their ability to interact and to inhibit factor VIIa/tissue factor: evidence for a two-step model of inhibition.

Inhibition of factor VIIa/tissue factor (TF) by extrinsic pathway inhibitor (EPI) requires the participation of factor Xa. Through this inhibition, factor Xa generated initially may feed back to suppress continuing generation of factor Xa via the extrinsic pathway during hemostasis. We have utilized chemical modifications of EPI and factor Xa to study the reactions responsible for inhibition. The data are consistent with a two-step model. First, EPI binds to factor Xa in a Ca2+ independent reaction in which the gla-domain of factor Xa does not participate. A functional active site on factor Xa and arginine residues on EPI are essential for this step. Then the factor Xa/EPI complex binds to factor VIIa/TF with resultant inhibition of its enzymatic activity. The gla-domain of factor Xa is essential for this step. Intact positively charged lysines on factor Xa may also be important.

Reliability of a modified tissue factor-dependent factor V assay for activated protein C resistant factor Va using a calcium-containing thromboplastin.

The original tissue factor-dependent factor V assay for activated protein C resistant factor Va (Blood 1995; 85: 1704-1711) has been modified to use a calcium containing thromboplastin and to express results as an observed to expected ratio (Obs/Exp.). The latter permits establishing a normal range independent of variations due to differences in reagents. Comparing Obs/Exp ratios with DNA analysis in 72 persons revealed that an Obs/Exp ratio of 0.6 distinguished without overlap normals from heterozygotes for FV R506Q. Three homozygotes had a ratio of < 0.1. Application of this Obs/Exp cut-off ratio of 0.6 to a total of 226 plasma samples tested to date discriminated without overlap between normals and heterozygotes. We conclude that this assay-readily adaptable to any dedicated coagulation laboratory and capable of yielding reliable results in all clinical circumstances in which testing is indicated-can distinguish between normals and heterozygotes for the FV R506Q mutation without the need for confirmatory DNA analysis.

Purification and properties of an abnormal blood coagulation factor IX (factor IXBm)/kinetics of its inhibition of factor X activation by factor VII and bovine tissue factor.

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

A plasma protein C activity assay suitable for a clinical laboratory. Its use to measure activity in hereditary and acquired deficiency states.

The authors describe the technic and standardization of an assay to measure plasma protein C activity feasible for clinical laboratory use. It is modified from an assay of Francis and Patch (Thromb Res 1983; 32:605-613) to enhance protein C recovery in the barium citrate eluate, to eliminate the steps of addition and neutralization of heparin, and to use only commercially available reagents. The normal range for plasma protein C in the assay is 72-130% (+/- 2 SD) (0.7-1.30). Hepatocellular disease lengthening the prothrombin time by 3-4 seconds was associated with plasma protein C activity of 25% (0.25) to 35% (0.35). Although the assay is thought to accurately measure protein C activity in patients taking warfarin, one cannot evaluate such patients for hereditary functional protein C deficiency because treatment with warfarin will itself reduce the ratio of protein C activity to antigen. The assay can be used in a patient receiving heparin if the heparin is removed as described.

A novel clotting assay for quantitation of plasma prothrombin (factor II) using Echis multisquamatus venom.

Measuring plasma prothrombin activity seems useful for evaluating thrombotic risk and managing oral anticoagulant therapy as an adjunct to the international normalized ratio. Therefore, we designed a new plasma prothrombin assay based on the ability of Echis multisquamatus venom to activate prothrombin with only calcium as a cofactor. In this assay, 1 part of undiluted citrated plasma is added to 5 parts of a venom reagent and the clotting time is measured. The assay's advantages are that dilution of the test plasma is required only when prothrombin activity exceeds 100%, a single standard curve can be used over months for a given batch of stock reagent, and barium-adsorbed plasma is used for dilution of test plasma and construction of the standard curve, thus eliminating the need for prothrombin-deficient plasma. However, one should be aware of the following: (1) test samples must contain at least 200 mg/dL fibrinogen; and (2) when prothrombin concentrations were below 50%, the venom-based assay often gave values up to 10% higher than the thromboplastin-based assay. Values obtained in 262 plasma samples tested with the venom-based assay and with a thromboplastin-based prothrombin assay correlated well (r2 = 0.93).

Clinical significance of antibodies to bovine and human thrombin and factor V after surgical use of bovine thrombin.

Two patients exposed during surgery to bovine thrombin developed antibodies reacting with both bovine and human thrombin and Factor V. Their thrombin times were markedly prolonged with bovine thrombin and modestly prolonged with human thrombin. High titer anti-bovine Factor V created diagnostic confusion in one patient by neutralizing bovine Factor V in a prothrombin assay substrate. Although weaker, antibody activity against human Factor V led to postoperative factor V deficiency in both patients. Such cross-reacting antibodies, recognizable by their higher titer against bovine than human Factor V, should be suspected when a patient surgically exposed to bovine thrombin develops a Factor V anticoagulant after operation. Crossed immunoelectrophoresis of adsorbed bovine plasma with each patient's plasma as antibody revealed many precipitin arcs indicative of immunization of the patients to additional proteins in a commercial thrombin preparation.