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The effects of gemfibrozil on several indices of haemostatic activity were explored in male patients with coronary heart disease (CHD). Sixty-three of 71 patients completed a crossover study in which gemfibrozil 1,200 mg/day and matching placebo were each taken in randomised order for 2 months in a double-blind manner, separated by a 2-month washout period. Serum cholesterol decreased by an average (95% confidence interval) of 12 (9 to 15)% and non-fasting triglyceride concentration by 43 (34 to 51)% during active treatment. Plasma prothrombin fragment F1 + 2 concentration, a marker of the in vivo rate of generation of thrombin, was 25 (12 to 37)% lower on average while on gemfibrozil than during the placebo phase. Factor VII coagulant activity (VIIc) and antigen concentration, and fibrinopeptide A concentration were not influenced by gemfibrozil in the group overall. However, the VIIc response appeared to be dependent upon the untreated cholesterol level. Hypercholesterolaemic men (cholesterol greater than 6.5 mmol/l) experienced a significant reduction in VIIc averaging 6% of standard during active therapy. Other effects of gemfibrozil were a 5 (2 to 9)% increase in plasma fibrinogen by a gravimetric method, an 11 (8 to 13)% increase in platelet count, and a 6 (2 to 10)% reduction in white cell count. The reduced incidence of CHD following gemfibrozil therapy in hyperlipidaemic patients may arise in part through a reduction in procoagulant activity and thus the risk of an occlusive coronary thrombosis.

We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.

Treatment with warfarin using a target International Normalized Ratio (INR) range of 1.7 to 2.5 is efficacious for many clinical indications, but the minimal intensity of anticoagulation required for antithrombotic protection has yet to be determined. To evaluate whether patients could be reliably monitored with a less intense regimen, we anticoagulated patients with warfarin for several months using a target INR range of 1.3 to 1.6 as determined by prothrombin time (PT) using a sensitive thromboplastin (Dade IS, International Sensitivity Index [ISI] = 1.3). Plasma measurements of F1+2, a marker of factor Xa action on prothrombin in vivo, were also obtained to determine the suppressive effect of warfarin on hemostatic system activity. Overall, 20 of 21 patients with a history of cerebrovascular events (mean age, 61 years) could be reliably regulated with warfarin in the target INR range. F1+2 levels were significantly suppressed from baseline in all patients, with a mean reduction of 49% (range, 28% to 78%). We found a significant relationship between the extent of suppression of prothrombin activation levels and the baseline measurements. A mean reduction of 65% was observed for those patients with baseline F1+2 greater than or equal to 1.5 nmol/L, but only 38% for baseline F1+2 less than or equal to 0.5 nmol/L. Overall, 68% of plasma samples obtained during stable anticoagulation were within the target INR range. PTs were also determined on all plasma samples with two thromboplastins of lower sensitivity (C+, ISI = 2.09; and automated simplastin, ISI = 2.10). Only 47% and 35% of PT determinations, respectively, were within the target range with these reagents. We conclude that prothrombin activation can be significantly suppressed in vivo with use of warfarin in an INR range of 1.3 to 1.6. This level of anticoagulation can be reliably achieved by monitoring PTs with a thromboplastin of high sensitivity.

Second malignancies are common in hairy cell leukemia. We report a case of a neuroendocrine carcinoma arising in a patient who had been diagnosed with hairy cell leukemia 6 years earlier. This case is the first report of these two tumors' occurring together. The pathogenetic basis for the presence of these two uncommon tumors in our patient is discussed.

Purer factor IX concentrates, containing very little or no factor II or X, have been developed in an attempt to avoid the thromboembolic complications that occur with prothrombin complex concentrates (PCC), which also contain factors II and X and variable amounts of factor VII. To evaluate ex vivo the thrombogenic potential of one of these purer concentrates, we studied whether large single doses produced signs of activation of the coagulation cascade in patients with haemophilia B, and compared the results with those obtained after infusion of a PCC. Seven patients were infused with 50 IU/kg of factor IX concentrate and seven additional patients were subsequently infused with 100 IU/kg of the same concentrate. After the infusions, factor IX levels rose in proportion to the administered dose while the concentrations of factor II and factor X did not rise at all. At both doses of concentrate, we did not observe significant post-infusion increments in the levels of the factor X activation peptide (a measure of the activity of the factor VIIa-tissue factor complex and/or the factor IXa-VIIIa-activated surface complex), prothrombin fragment 1 + 2 (a measure of factor Xa activity), and fibrinopeptide A (a measure of thrombin activity). We also infused 10 patients with a PCC (50 IU/kg). After the infusions, significant rises in the concentrations of the factor X activation peptide and prothrombin fragment were observed. Therefore, it appears that the infusion of a PCC to patients with haemophilia B can augment factor X activation and subsequently thrombin generation in vivo and that this process can be abrogated by the administration of more pure factor IX concentrate.

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.

The levels of prothrombin fragment F1 + 2 were measured by a double antibody radioimmunoassay in blood samples collected into different anticoagulant solutions. We evaluated healthy males between the ages of 42 and 77, asymptomatic patients with hereditary deficiencies of protein C or protein S, and persons receiving tumor necrosis factor infusions. The results in specimens collected in an anticoagulant containing ACD, EDTA, adenosine, and 25 U/ml of heparin (a) were highly correlated with those collected in an anticoagulant containing a synthetic thrombin inhibitor, EDTA, and aprotinin (b). However, in asymptomatic patients with congenital antithrombin III deficiency, we found that the plasma levels of F1 + 2 in blood collected in anticoagulant (a) were usually substantially higher than those collected in anticoagulant (b). We determined that this phenomenon was not attributable to the venipuncture procedure itself, but rather appears to be due to the action of low concentrations of heparin in the presence of reduced blood levels of antithrombin III. Our data show that the previously documented elevations in plasma F1 + 2 levels in patients with congenital antithrombin III deficiency appear to be caused by the above in vitro anticoagulant effect, and that this population does not exhibit evidence of a prethrombotic state as defined by the F1 + 2 assay.

Prothrombin complex concentrates (PCC), licensed for the treatment of hemophilia B, are known to carry a significant risk of thromboembolic complications. Although the reasons for thrombogenicity are not completely understood, several manufacturers have developed purified factor IX concentrates that contain negligible amounts of the other vitamin K-dependent factors. To evaluate whether or not the infusion of such a factor IX concentrate is followed by lesser activation of the hemostatic system than by the infusion of a PCC, we performed a series of coagulation assays on 11 hemophilia B patients before and after the administration of these two types of concentrate using a randomized cross-over design. The levels of prothrombin fragment F1 + 2, a sensitive measure of the in vivo cleavage of prothrombin by factor Xa, was significantly increased in plasma after PCC, but not after factor IX concentrate. Plasma fibrinopeptide A, a sensitive index of the enzymatic activity of thrombin on fibrinogen, also increased significantly after PCC but not after factor IX concentrate. The fragment B beta 15-42, a sensitive index of the enzymatic action of plasmin on fibrin II, did not change after either concentrate. There were also no differences in less sensitive coagulation measurements, such as plasma fibrinogen, antithrombin III, and fibrin monomers, nor in indices of platelet activation, such as beta-thromboglobulin and platelet factor 4. These findings show that the infusion of a purified factor IX concentrate can result in substantially less activation of the coagulation cascade than may be seen with PCC.

Several investigators have reported that tumor necrosis factor (TNF) can alter the production of plasminogen activator type-1 (PAI-1) and plasminogen activators (PAs) by endothelial cells in vitro. We have examined the in vivo effects of recombinant human TNF administration on fibrinolysis as assessed by parameters in plasma during a 24-hour period of continuous TNF infusion to 17 cancer patients with active disease. The plasma levels of PAI activity increased sevenfold after 3 and 24 hours of TNF infusion. This was the result of an increase of PAI-1 antigen; PAI-2 antigen was not detectable. Plasma concentrations of tissue-type PA (t-PA) antigen increased twofold to fivefold after 3 and 24 hours of TNF infusion, whereas urokinase-type PA antigen levels in plasma remained unaltered. After 3 hours of TNF infusion the plasma levels of alpha 2-antiplasmin were slightly decreased, 5% on average, suggesting that fibrinolysis continued. After 24 hours of TNF infusion a highly significant increase in fibrin- plus fibrinogen-degradation products, and separately of fibrin degradation products and fibrinogen degradation products, was found. This indicates that fibrinolysis persisted, at least partly, in the presence of high levels of PAI activity. Whereas PAI-1 production increased, t-PA production by human endothelial cells in vitro remains unaltered or even decreases on TNF addition. It has been shown previously that TNF infusion in our patients results in thrombin and fibrin generation. Therefore, it is possible that thrombin, not TNF, is the actual stimulus for t-PA production in our patients. We speculate that fibrin is formed during TNF infusions and that plasmin is generated by t-PA action immediately on the initial formation of (soluble) fibrin molecules. Such a process may explain the generation of degradation products of both fibrin and fibrinogen during infusion of TNF in patients.

In experimental animals, conditions which drastically decrease cardiac output may reduce the strength and endurance of respiratory muscles leading to hypercapnic respiratory failure. Because patients with chronic CHF have reduced cardiac output and vital capacity (FVC), we measured PImax and PEmax and maximal handgrip force in 16 patients with CHF and 18 AMNs. The patients with CHF had a mean left ventricular ejection fraction of 26 +/- 7 percent. Maximal respiratory pressures were significantly reduced; group mean values (+/- SD) for PImax at FRC were 41.4 +/- 5.6 cm H2O (CHF) and 102.1 +/- 27.4 cm H2O (AMN) (p less than 0.001), with PImax values in five patients with CHF as low as 20 to 30 cm H2O. In most patients, PEmax was comparably reduced. Handgrip force was less dramatically reduced, suggesting selective respiratory muscle weakness. Possible explanations include reduction in respiratory muscle blood flow or generalized muscular atrophy and weakness related to cardiac cachexia.

Despite significant progress in elucidating the biochemistry of the hemostatic mechanism, the process of blood coagulation in vivo remains poorly understood. Factor IX is a vitamin K-dependent glycoprotein that can be activated by factor XIa or the factor VII-tissue factor complex in vitro. To investigate the role of these two pathways in factor IX activation in humans, we have developed a sensitive procedure for quantifying the peptide that is liberated with the generation of factor IXa. The antibody population used for the immunoassay was raised in rabbits and chromatographed on a factor IX-agarose immunoadsorbent to obtain antibody populations with minimal intrinsic reactivity toward factor IX. We determined that the mean level of the factor IX activation peptide (FIXP) in normal individuals under the age of 40 years was 203 pmol/L and that levels increased significantly with advancing age. The mean concentration of FIXP was markedly reduced to 22.7 pmol/L in nine patients with hereditary factor VII deficiency (factor VII coagulant activity less than 7%) but was not significantly different from normal controls in nine subjects with factor XI deficiency (factor XI coagulant activity less than 8%). These data indicate that factor IXa generation in vivo results mainly from the activity of the tissue factor mechanism rather than the contact system (factor XII, prekallikrein, high molecular-weight kininogen, factor XI). Our results may also help to explain the absence of a bleeding diathesis in many patients with deficiencies of the contact factors of coagulation.

Tumor necrosis factor has been implicated in the activation of blood coagulation in septicemia, a condition commonly associated with intravascular coagulation and disturbances of hemostasis. To evaluate the early dynamics and the route of the in vivo coagulative response to tumor necrosis factor, we performed a controlled study in six healthy men, monitoring the activation of the common and intrinsic pathways of coagulation with highly sensitive and specific radioimmunoassays. Recombinant human tumor necrosis factor, administered as an intravenous bolus injection (50 micrograms per square meter of body-surface area), induced an early and short-lived rise in circulating levels of the activation peptide of factor X, reaching maximal values after 30 to 45 minutes (mean +/- SEM increase after 45 minutes, 34.2 +/- 18.2 percent; tumor necrosis factor vs. saline, P = 0.015). This was followed by a gradual and prolonged increase in the plasma concentration of the prothrombin fragment F1+2, peaking after four to five hours (mean increase after five hours, 348.0 +/- 144.8 percent; tumor necrosis factor vs. saline, P less than 0.0001). These findings signify the formation of factor Xa (activated factor X) and the activation of prothrombin. Activation of the intrinsic pathway could not be detected by a series of measurements of the plasma levels of factor XII, prekallikrein, factor XIIa-C1 inhibitor complexes, kallikrein-C1 inhibitor complexes, and the activation peptide of factor IX. The delay between the maximal activation of factor X and that of prothrombin amounted to several hours, indicating that neutralization of factor Xa activity was slow. We conclude that a single injection of tumor necrosis factor elicits a rapid and sustained activation of the common pathway of coagulation, probably induced through the extrinsic route. Our results suggest that tumor necrosis factor could play an important part in the early activation of the hemostatic mechanism in septicemia.

A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL promoter. In flask cultures at lower cell density, the mutant again produced less acetate than the parent, although both showed a much lower level of acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression level of IL-2 in flask cultures, and there was a greater difference between the mutant and its parent in the final extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentration of acetate in the medium, which was much higher in fermentor cultures (greater than or equal to 300 mM after 5 h of induction) than in flask cultures (less than or equal to mM) of the parent organism, was a significant factor in limiting expression of the heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not slow growth.

Six patients with disseminated intravascular coagulation (DIC) in association with acute promyelocytic leukemia (APL) were studied with sensitive radioimmunoassays that are able to quantitate the extent of thrombin generation within the human circulation. The levels of prothrombin activation fragment, F1 + 2, and thrombin-antithrombin complex (TAT) were obtained at clinical presentation and were then followed serially in several patients during induction chemotherapy. The antileukemic therapy often resulted in a rise in the plasma levels of these molecular species. Simultaneous measurements of fibrinopeptide A (FPA) were also obtained, and the concentrations of this polypeptide were correlated with the levels of F1 + 2 and TAT in patients who were not receiving heparin. Nine individuals with other morphological subtypes of acute nonlymphocytic leukemia (ANLL) were investigated and were usually found to have increased levels of F1 + 2, TAT, and FPA at clinical presentation. However, the magnitude of the elevations was considerably greater and the correlation between TAT and FPA levels was stronger in APL than in ANLL. These studies provide direct evidence that patients with APL, as well as ANLL, generate excessive amounts of thrombin within their vascular system. Furthermore, the data suggest that the concentrations of F1 + 2, compared with the levels of FPA, may be a more sensitive indicator of hemostatic system hyperactivity in individuals with DIC.

We have evaluated the efficacy of utilizing radioimmunoassays (RIAs) for prothrombin activation fragments (F2/F1 + 2) and for thrombin--antithrombin complex (TAT) in purified systems and in whole blood. During venipuncture, appropriate anticoagulants were employed in order to prevent the generation of thrombin and factor Xa. The RIAs were shown to be specific for F2/F1 + 2 as well as TAT and did not interact with other plasma components. Initially, thrombin generation was studied in a purified human system of prothrombin, antithrombin, factor Xa, and factor V as well as phospholipid and Ca++. Under these conditions, the kinetics of F2/F1 + 2 and TAT generation were virtually superimposable. However, when factor V was omitted from the reaction mixture, a significantly greater amount of F2/F1 + 2 as compared to TAT was observable. Subsequently, prothrombin activation was monitored during the spontaneous coagulation of freshly drawn blood. Throughout the entire course of thrombin generation, the observable rate of formation of F2/F1 + 2 was considerably greater than that of TAT. We have examined the levels of F2/F1 + 2 and TAT in normal individuals. Our studies indicate that the concentrations of F1 + 2 and TAT average 1.97 nM and 2.32 nM, respectively. We have also quantitated the concentrations of F2/F1 + 2 and TAT in patients with disseminated intravascular coagulation. In these individuals, the levels of both components are elevated. However, the ratio of F1 + 2 to TAT ranges from 2.37 to 5.55. Thus, we conclude that under in vivo conditions, prothrombin activation is characterized by the accumulation of a stable precursor, such as prethrombin-2, and that this phenomenon may be related to an alteration of factor V function.

We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.

The partial characterization of a dysfunctional protein C (PC), provisionally named "PC Cádiz", in a 45-year-old male patient suffering from recurrent venous thrombosis is described. The only defect found in laboratory assays for haemostasis and hepatic function was a half normal level of both amidolytic and anticoagulant protein C activity, measured by different functional assays that use thrombin-thrombomodulin complex and a snake venom to activate protein C. Protein C antigen was always found to be within normal levels. Two young daughters of the propositus were found to have the same defect. Double-crossed immunoelectrophoresis, performed in the presence and absence of Ca2+ in the first dimension, showed no clear differences between patient and control PC. PC adsorption to barium salts was also found to be normal. Measurement of the PC activation peptide in the barium citrate eluates after PC activation showed no significant differences between patient and 10 normal controls, the concentration of this peptide being very similar to that of PC zymogen in the same eluates before PC activation. These results indicate that this abnormal PC is able to be normally activated by thrombin-thrombomodulin complex but does not exhibit serine protease activity, probably due to a defect in the PC molecule near the active site center.

Factor VIII antigen (VIII:CAg) exhibits molecular weight heterogeneity in normal plasma. We have compared the relative quantities of VIII:CAg forms present in normal individuals (n = 22) with VIII:CAg forms in renal dysfunction patients (n = 19) and in patients with disseminated intravascular coagulation (DIC; n = 7). In normal plasma, the predominant VIII: CAg form, detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was of molecular weight 2.4 X 10(5), with minor forms ranging from 8 X 10(4) to 2.6 X 10(5) D. A high proportion of VIII:CAg in renal dysfunction patients, in contrast, was of 1 X 10(5) mol wt. The patients' high 1 X 10(5) mol wt VIII: CAg level correlated with increased concentrations of serum creatinine, F1+2 (a polypeptide released upon prothrombin activation), and with von Willebrand factor. Despite the high proportion of the 1 X 10(5) mol wt VIII:CAg form, which suggests VIII:CAg proteolysis, the ratio of Factor VIII coagulant activity to total VIII:CAg concentration was normal in renal dysfunction patients. These results could be simulated in vitro by thrombin treatment of normal plasma, which yielded similar VIII:CAg gel patterns and Factor VIII coagulant activity to antigen ratios. DIC patients with high F1+2 levels but no evidence of renal dysfunction had an VIII:CAg gel pattern distinct from renal dysfunction patients. DIC patients had elevated concentrations of both the 1 X 10(5) and 8 X 10(4) mol wt VIII:CAg forms. We conclude that an increase in a particular VIII:CAg form correlates with the severity of renal dysfunction. The antigen abnormality may be the result of VIII:CAg proteolysis by a thrombinlike enzyme and/or prolonged retention of proteolyzed VIII:CAg fragments.

A family is described in which venous thrombosis developed in five members as early as 14 years of age. Routine coagulation studies, plasma antithrombin III, factor V, plasminogen, beta-thromboglobulin, fibrinopeptide A, prothrombin fragment F1+2, and thrombin-antithrombin III complex were all within normal limits. However, defective release of vascular plasminogen activator was observed on several occasions in all five subjects as compared with a control population of 125 persons (0.04 Committee on Thrombolytic Agents [CTA] units/ml plasma as compared with 0.21 CTS units/ml). In addition, levels of factor VII/von Willebrand's factor were significantly elevated above the normal range in this pedigree.

Inherited antithrombin III deficiency is associated with an increased risk of thromboembolism. Using recombinant-DNA techniques, we isolated a molecular probe for the antithrombin III structural gene and identified a common DNA polymorphism within the gene. We found that there is genetic heterogeneity in this disorder. In one family, the antithrombin III gene was deleted in affected members, whereas in another no deletion occurred. Use of the DNA polymorphism should allow identification and further characterization of abnormal antithrombin III genes.

The presence of hereditary protein C deficiency has been shown to predispose patients to the development of venous thrombosis. We used radioimmunoassays for the protein C activation peptide (PCP) and the prothrombin fragment F1 + 2 to quantitate the extent of in vivo activation of protein C by thrombin-thrombomodulin and prothrombin by factor Xa, respectively, in the blood of individuals with this clinical disorder. A total of 46 protein C deficient subjects from 18 kindreds were studied. In 23 nonanticoagulated patients with an isolated deficiency of protein C, the mean level of PCP was substantially reduced while the mean concentration of F1 + 2 was significantly elevated as compared with normal controls (1.10 pmol/L v 1.78 pmol/L, P less than .0005 and 2.54 nmol/L v 1.51 nmol/L, P less than .0005, respectively). The metabolic behavior of 131I-F1 + 2 was found to be similar in protein C deficient patients and normal individuals. However, we were unable to establish a significant correlation between decreased PCP levels and increased F1 + 2 measurements in these 23 patients. This study demonstrates that heterozygous protein C deficient individuals with equivalent plasma levels of the zymogen may have markedly different biochemical profiles when assay techniques are used that quantitate the in vivo activity of the coagulation system. Six individuals from three pedigrees were identified as having combined deficiencies of protein C and either antithrombin III or protein S; the genetic basis for the combined deficiency state was determined in two of the kindreds. Finally we observed that hemostatic system activity as measured by the PCP and F1 + 2 assays is markedly suppressed in protein C deficient patients who are chronically anticoagulated with coumarin derivatives.

A family with a high incidence of spontaneous thromboembolism over four generations has been investigated. The propositus is a 21-yr-old male with a history of thrombophlebitis. Medical histories of 46 family members were obtained. Twelve of these individuals have experienced deep venous thromboses and/or pulmonary emboli. Seven members of the kindred, with a prior history of thrombotic phenomena, were investigated in detail. These subjects were found to have normal plasma concentrations of immunoreactive antithrombin (mean 96%), decreased plasma levels of progressive antithrombin activity (mean 50%), and greatly reduced amounts of plasma heparin cofactor activity (mean 42%). The abnormal antithrombin ("Chicago") was found to elute from heparin-Sepharose at a higher ionic strength than normal inhibitor. The functionally defective antithrombin molecules exhibit a reduced ability to neutralize thrombin in the presence or absence of heparin (approximately 10%-20% of normal). The molecular defect of this protease inhibitor thus appears to be distinct from those of previously described abnormal antithrombins.

The coagulation cascade can be pictured as a series of reactions in which a zymogen, a cofactor, and a converting enzyme interact to form a multimolecular complex on a natural surface. In each case, the four reactants must be present if the conversion of a zymogen to the corresponding serine protease is to take place at any significant rate. The principal natural anticoagulant systems that are able to exert damping effects on the various steps of the cascade are the heparin-antithrombin and protein C-thrombomodulin mechanisms that regulate the serine proteases and the cofactors or activated cofactors, respectively. Inherited thrombotic disorders associated with specific deficiencies of antithrombin, protein C, and protein S have been described. This review describes the biochemistry and physiology of these endogenous anticoagulant systems. The development of specific radioimmunoassay techniques for prothrombin activation fragment F1 + 2, fibrinopeptide A, and protein C activation peptide has allowed us to carry out studies of these endogenous regulatory mechanisms involved in thrombin generation in patients with deficiencies of antithrombin or protein C. This information is then used to construct a framework for understanding the pathophysiology of the prethrombotic and actively thrombotic states in humans with these clinical disorders.

We have developed a radioimmunoassay (RIA) for prethrombin 2 (Pr2), a potential intermediate in the transformation of prothrombin to thrombin. Antisera against human Pr2 were raised in rabbits and the respective immunoglobulin G fractions were chromatographed on prothrombin-Sepharose. The specific antibody population obtained was used to construct a double-antibody RIA capable of measuring as little as 0.05 nmol/L of this component. The immunoreactivity of prothrombin was approximately 40,000 times less than that of Pr2 on a molar basis. Because of nonspecific contributions of plasma constituents to the immunoreactive signal, the measurement of Pr2 in this milieu required the use of a titration curve in which Pr2 was added back to Pr2-depleted plasma. This assay was then used to determine the levels of this species in two patient populations with increased prothrombin activation as determined by the prothrombin fragment F1 + 2 RIA, a measure of the in vivo cleavage of prothrombin by factor Xa. The mean Pr2 concentrations in eight patients with disseminated intravascular coagulation and six asymptomatic individuals with congenital antithrombin deficiency not receiving antithrombotic therapy were not significantly elevated as compared with those of normal controls (0.244 nmmol/L and 0.242 nmol/L vs. 0.184 nmol/L, respectively). Our studies show that Pr2 is cleared from the plasma of dogs with a t1/2 of approximately 25 minutes. Given that the t1/2 of F1 + 2 is estimated to be approximately 90 minutes, the low plasma levels of Pr2 observed in patients with thrombophilia cannot result from rapid clearance of this component.(ABSTRACT TRUNCATED AT 250 WORDS)

Several investigators have reported that tumor necrosis factor (TNF) can alter the hemostatic properties of vascular endothelial cells in vitro. We have examined the in vivo effects on the hemostatic mechanism of recombinant human TNF administered as a continuous intravenous infusion to 23 cancer patients with active disease. A battery of sensitive and specific immunochemical techniques were used to monitor changes in blood coagulability. Serial determinations of F1 + 2, the protein C activation peptide (PCP), and fibrinopeptide A (FPA) were obtained prior to the initiation of the TNF infusions and at three and 24 hours after the start of therapy in 12 individuals who received greater than 3 x 10(5) U/m2/24h. The mean levels of F1 + 2, PCP, and FPA were significantly elevated at both time points as compared to the baseline values. The metabolic behavior of 125I-F1 + 2 in an animal model was not affected by infusions of the cytokine. We therefore conclude that the observed elevations in the concentration of this marker in humans receiving TNF result from hemostatic system hyperactivity. In 11 subjects infused with 1 x 10(5) to 2.4 x 10(5) U/m2/24 h of the cytokine, the mean levels of F1 + 2, PCP, and FPA were not significantly greater at 24 hours as compared with the baseline values, indicating that there is a threshold dose at which the cytokine can exert a biochemical effect on the coagulation system. Our studies demonstrate that TNF is able to provide a substantial net procoagulant stimulus to the hemostatic mechanism, and suggest that this cytokine may be a mediator of certain hypercoagulable states in humans.

We have studied tissue factor gene expression in the leukocytes of 22 patients with acute myeloblastic leukemia (AML). Total RNA from peripheral blood or bone marrow cells depleted of monocytes was analyzed by Northern blot analysis using a 32P-labeled tissue factor cDNA probe. Cells from 10 cases expressed tissue factor mRNA and positive cases were distributed among the myeloblastic, myelomonocytic, and monocytic subtypes of AML. Tissue factor transcripts were not detected in cells derived from normal bone marrow. The expression of this gene product on the surface of leukemic cells could contribute to the excessive thrombin generation that has been observed in some individuals with this disorder.

In view of the known association of vascular disease with increasing age, we have conducted an analysis of hemostatic system activity with respect to perturbations induced by aging phenomena. We have utilized an immunochemical assay for prothrombin fragment F1 + 2 to quantify Factor Xa activity upon prothrombin in the plasma of 199 healthy males between the ages of 42 and 80. The levels of F1 + 2 in this population generally increased as a function of age (P less than 0.0001). The metabolic behavior of this marker was determined in 10 individuals greater than 65 yr of age with varying levels of F1 + 2, which ranged from 1.28 to 5.85 nM. The elevations in the concentration of this component were not due to diminished clearance of the fragment. Radio-immunoassays for fibrinopeptide A (FPA) and the protein C activation peptide (PCP) were subsequently employed to measure thrombin activity upon fibrinogen and thrombin-thrombomodulin activity upon protein C, respectively, in 82 members of this population ranging in age from 42 to 80. Significant positive correlations were again observed between increasing age and the level of F1 + 2 (P less than 0.0001) as well as FPA (P less than 0.01) and PCP (P less than 0.002). The results of this cross-sectional study indicate that many apparently normal males of increasing age with normal immunologic levels of antithrombin III and protein C exhibit a biochemical defect that denotes the presence of an acquired prethrombotic state.

Heparin was covalently bound to solid substrate surfaces by means of four different chemistries. It was coupled to polymethylacrylate (PMA) beads with glutaraldehyde, carbodiimide, or radical polymerization initiated by Ce4+, or to agarose beads with cyanogen bromide. Each of these chemistries produced measurable amounts of surface-bound heparin, which was minimally elutable in contact with plasma. Antithrombin (AT) binding by heparinized PMA materials (compared with PMA control beads) ranged from no AT binding for the material heparinized with carbodiimide (PMA-Alb-Hep(EDC] to 3.6 micrograms/ml packed beads for the material heparinized by radical polymerization (PMA-MA-Hep). Heparin-like catalytic activity of these materials (assayed by measuring the generation of thrombin-antithrombin complex in plasma) correlated well with the amount of heparin bound, but not as well with AT binding capacity. Heparinized agarose, which exhibited a large AT binding capacity (2.2 mg AT per milliliter of packed gel), had virtually no catalytic activity because of its inability to release thrombin-antithrombin complex from the surface. Platelet interaction with heparinized materials that exhibit high AT binding capacity was reduced by pretreatment with normal plasma but not by pretreatment with AT-depleted plasma. Platelet interaction with heparinized materials with low AT binding capacities was not reduced by pretreatment with normal plasma. We conclude that AT binding by heparin reduces the platelet reactivity of heparinized surfaces.

RIAs for hemostatic system activation were employed to study patients who were anticoagulated with warfarin. The mean prothrombin fragment F1 + 2 concentration in stably anticoagulated individuals without an inherited thrombotic diathesis (mean prothrombin time [PT] ratio [PT of patient/PT of normal plasma pool] = 1.74) was 0.231 nM as compared with a mean plasma F1 + 2 level of 1.68 nM for a nonanticoagulated control group (P less than 0.0001). The initiation of oral anticoagulants in two subjects who did not exhibit protein C deficiency led to a paradoxical increase in F1 + 2 levels during the first day of therapy. We have also shown that a relatively low intensity regimen of warfarin (PT ratio less than 1.2) may reduce elevated concentrations of F1 + 2 into the normal range in patients with a history of recurrent thromboembolism. The mean F1 + 2 level in antithrombin-deficient individuals on warfarin was significantly elevated (mean = 0.714 nM) as compared with that in anticoagulated subjects with protein C deficiency (mean = 0.205 nM) or in those without an inherited thrombotic disorder (P less than 0.01) at equivalent levels of intensity of oral anticoagulation. We therefore conclude that the effect of warfarin on hemostatic system activation is modulated by the endogenous heparan sulfate-antithrombin mechanism.

L-asparaginase, a chemotherapeutic agent employed in the treatment of acute lymphocytic leukemia (ALL), is known to depress the synthesis of numerous plasma proteins. The plasma concentration of the major protease inhibitor of the coagulation mechanism, antithrombin III, is substantially decreased in patients receiving this drug. This observation has generated speculation that L-asparaginase may induce a hypercoagulable state in humans. To examine this hypothesis, we studied ten patients with ALL in remission who were being treated with the above chemotherapeutic agent. Our data revealed that infusion of this enzyme leads to a marked decrease in the plasma concentrations of prothrombin as well as antithrombin III. However, we have also observed a constant level of thrombin generation during the same period of time as monitored by plasma levels of prothrombin activation fragment (F1 + 2) and thrombin-antithrombin complex (TAT). Based upon these findings we suggest that administration of L-asparaginase does not usually lead to the induction of a hypercoagulable state.

Antithrombin III (ATIII) has been expressed in transiently transfected COS-1 monkey cells and in stably transformed Chinese hamster ovary cells, and the resultant protein has been characterized for biological activity. Both cell types efficiently secrete high levels of heterogeneous molecular weight forms of ATIII antigen. The heterogeneity results from differences in post-translational modifications. However, only a small percentage (5-10%) of the total antigen expressed is biologically active. The fraction of biologically active ATIII has been purified from total ATIII by affinity fractionation on heparin-Sepharose. This fractionation indicates that the differences in the active and inactive forms of expressed ATIII result from differences in their ability to bind heparin. Purified ATIII has a specific activity very similar to that of plasma-derived ATIII and exhibits typical heparin-accelerated ATIII activity. The biologically active fraction of ATIII appears to represent the higher molecular weight forms of the ATIII expressed and is likely not a result of altered asparagine-linked glycosylation; however, the nature of the post-translational modification required for ATIII activity remains unclear. The ability to express biologically active ATIII at such high levels should allow further investigations of the structural requirements for ATIII activity.

Hereditary antithrombin III (ATIII) deficiency predisposes patients to venous thrombosis. The prothrombin fragment F1+2 radioimmunoassay demonstrates that many asymptomatic patients with this disorder not receiving antithrombotic therapy have elevated plasma factor Xa activity. The hemostatic system hyperactivity as measured by this assay could be specifically corrected by rising plasma ATIII levels of several persons into the normal range. This indicates that the prethrombotic state can be defined as an imbalance between the production and inhibition of factor Xa enzymatic activity. The effects of warfarin on factor Xa enzymatic activity in persons with congenital ATIII deficiency have also been evaluated. At equivalent intensities of oral anticoagulation, the mean plasma F1+2 level in patients with ATIII deficiency was significantly elevated as compared with anticoagulated persons without this inherited thrombotic disorder. It is concluded that the effect of warfarin on hemostatic system activation is modulated by the endogenous heparan sulfate-ATIII mechanism. This suggests that the F1+2 radioimmunoassay can be employed to improve the understanding of the hypercoagulable state associated with antithrombin III deficiency as well as to develop more effective treatment strategies to prevent thromboembolic events in patients with this disorder.

Twenty patients with pulmonary complications associated with combination chemotherapy regimens containing bleomycin were studied to determine common patterns of pulmonary radiographic abnormalities. All patients were receiving bleomycin on one of two different regimens of combination chemotherapy. Ten patients with non-Hodgkin lymphoma received a relatively low dose (22-64 mg total) and 10 patients with testicular cancer received a higher dose (360 mg total). The high-dose group showed subclinical radiographic lung toxicity changes in eight (80%) patients during and after therapy. Chronic pulmonary abnormalities were seen in nine (90%) patients in the high-dose group but in only three (30%) patients in the low-dose group. These was no significant difference in the pattern and distribution of lung infiltrates in these two groups. Infiltrates involving mainly the costophrenic triangle were seen in 18 (90%) patients; in six (33%) of these the changes were confined to the costophrenic triangles bilaterally and nearly symmetrically. Five (25%) patients had infiltrates at the periphery of the lungs. Elevation of the diaphragm was seen in 16 (80%) patients. A pleural reaction without gross effusion was seen in nine (45%) patients, of whom five (55%) demonstrated thickening of the interlobar fissures. Chronic lung changes were mostly confined to the bases in the form of failure of reexpansion of the costophrenic triangle (55%), persistent elevation of the diaphragm (35%), and a reticular meshwork of fibrosis at the costophrenic triangles (25%). Minimal lung disease was manifest as ground glass appearance at the lung bases and as fine, linear or reticulonodular densities that involved the costophrenic triangles.

The presence of congenital antithrombin deficiency has been consistently shown to predispose patients to venous thrombosis. We have utilized the prothrombin fragment F1+2 radioimmunoassay to quantitate factor Xa activity in the blood of 22 asymptomatic individuals with this clinical disorder not receiving antithrombotic therapy. The mean level of F1+2 was significantly elevated in these patients as compared to normal controls (3.91 vs. 1.97 nM, P less than 0.001). The metabolic behavior of 131 I-F1+2 was found to be similar in antithrombin-deficient subjects and normal individuals. The hemostatic system hyperactivity as measured by the F1+2 assay could be specifically corrected by raising the plasma antithrombin levels of the above asymptomatic individuals into the normal range. This study provides the first demonstration that the prethrombotic state can be biochemically defined as an imbalance between the production and inhibition of factor Xa enzymatic activity within the human circulation. It is known that antithrombin and alpha 1-proteinase inhibitor (PI) are the major inhibitors of factor Xa in human plasma in the absence of heparin. To further evaluate the mechanism by which antithrombin functions as an inhibitor of factor Xa in humans, we studied five patients who exhibited severe congenital deficiencies of alpha 1-PI. Our results indicated that the plasma of these subjects showed virtually identical decreases in plasma antifactor Xa activity in the absence of heparin when compared to antithrombin-deficient individuals, but the plasma F1+2 levels in the alpha 1-PI deficient population were not significantly different than normal. This data suggests that alpha 1-PI does not function as a major inhibitor of factor Xa in vivo, and that a tonically active heparin-dependent mechanism exists in humans for accelerating the neutralization of this enzyme by antithrombin.

Protein C is an important anticoagulant in circulating blood. The balance between the procoagulant and anticoagulant pathways determines whether a patient is at risk for thrombosis or hemorrhage, and low levels of protein C may result in thrombotic disease. It is important for clinical coagulation laboratories to measure protein C. For this purpose, a simple functional assay is needed. This article describes an automated assay for functional protein C with activation by copperhead venom. The authors have automated this assay for a centrifugal analyzer (COBAS Bio) and have proved the specificity of the assay by comparing values obtained amidolytically with those obtained with a radioimmunoassay for protein C.

A family with a high incidence of venous thromboembolism was investigated. We performed medical evaluations on 184 of the 411 surviving members of the pedigree, which allowed assignment of individuals into positive, equivocal, or negative categories with respect to their clinical histories of thrombosis. Subjects with antigenic levels of protein C less than 66% of a normal plasma pool were classified as having protein C deficiency. Positive thrombotic histories were found in 13 of the 46 family members determined to be protein-C deficient and in only five of their 138 biochemically unaffected relatives. Statistical analysis of the association between thromboembolic disease and protein-C deficiency was strongly positive chi 2 = 24.95, P less than .0001 with n = 184), indicating that heterozygous protein-C deficiency is an important independent risk factor for the development of thrombotic manifestations in this pedigree. However, the absence of thromboembolic manifestations in many of the protein-C deficient family members to date indicates that other, as yet undefined, factors must play an important role in the clinical expression of this disorder.

Numerous investigators have postulated that a hypercoagulable state exists in humans for a period of time before the development of thrombotic episodes. A clear biochemical definition of the prethrombotic state, however, has proved elusive due in part to the lack of reliable techniques for monitoring pertinent changes in blood coagulability. Based on recent advances in our knowledge of the biochemistry of the coagulation system, a series of highly sensitive and specific immunochemical tools has been developed that can quantitate the activities of various steps of the hemostatic mechanism in vivo at the subnanomolar level. We have established assays for F1+2 and the protein C activation peptide, which measure the cleavage of the prothrombin molecule by factor Xa and the scission of protein C by the thrombin-thrombomodulin complex, respectively. Nossel and coworkers had previously constructed similar assays for fibrinopeptide A (FPA) and fragment B beta 1-42, which monitor the cleavage of fibrinogen by thrombin and the proteolysis of fibrin I by plasmin, respectively. Substantial elevations in the levels of these markers have been found in patients with disseminated intravascular coagulation and many subjects with acute deep venous thrombosis. The F1+2 and FPA assays have been used to demonstrate that significant increments in factor Xa activity but not thrombin activity regularly occur in the blood of nonanticoagulated individuals with congenital deficiencies of antithrombin or protein C. These two disorders are known to be correlated with the subsequent development of thrombosis. Patients with protein C deficiency have also been noted to have significantly reduced plasma levels of protein C activation peptide. By using the immunoassays for FPA and B beta 1-42 in studies of postoperative patients, it has been shown that an imbalance between the procoagulant action of thrombin and the anticoagulant effect of plasmin on fibrin I polymer may induce an acquired thrombotic diathesis. Finally, we have recently demonstrated that prothrombin activation as measured by the F1+2 assay is suppressed by oral anticoagulants in the blood of patients with thrombotic diatheses. These investigations suggest that these assay techniques can be used to improve our understanding of the hypercoagulable state as well as to develop more effective treatment strategies for the prevention of thromboembolic events.

Two cases of acute aortic thrombosis, a previously unreported complication of antithrombin III deficiency, are reported. Both patients had abnormally low antithrombin III levels, which improved to normal levels with warfarin ;sodium therapy. The possibility of antithrombin III deficiency should be considered in young patients with acute arterial thrombosis.

Phase I clinical studies of antithrombin III (ATIII) concentrate demonstrated a mean in vivo incremental recovery of functional activity of 1.4 percent per unit/kg administered, an initial 50 percent disappearance time of 22 hours, and a biologic half-life of 3.8 days. Based on these observations, a treatment regimen designed to maintain plasma ATIII levels between 75 and 120 percent of normal has been developed. None of 10 subjects with congenital ATIII deficiency treated prophylactically had evidence of thromboembolism, including four pregnant women at the time of delivery. Five subjects treated for acute thrombosis and/or thromboembolism, four of whom were pregnant, recovered without further thrombotic extension or recurrence. Heparin resistance was reversed in two subjects, both pregnant. Nine subjects with acquired ATIII deficiency also received ATIII treatment for venous or arterial thrombosis or disseminated intravascular coagulation, all with low plasma ATIII levels. Two subjects with disseminated intravascular coagulation demonstrated improvement, one clinically, the other biochemically. All patients with congenital ATIII deficiency survived, but only five of nine with acquired deficiency survived, highlighting the importance in acquired ATIII deficiency of the underlying disease in prognosis. Survival rate was especially poor in subjects with arterial thrombosis in the setting of low plasma ATIII. Administration of ATIII concentrate was well tolerated. None of the subjects who received ATIII concentrate demonstrated evidence of an infectious transmissible agent. These studies demonstrate that it is now feasible to safely replace the deficient protein in congenital ATIII deficiency, either prophylactically or therapeutically.

A sensitive radioimmunoassay (RIA) for the fragment that is liberated from factor X when this zymogen is activated by factor VII/VIIa-tissue factor or factor IXa was developed. Antisera were raised in rabbits to a synthetic 15 amino acid peptide containing the COOH-terminal sequence of the activation fragment coupled to bovine serum albumin with glutaraldehyde. The reactivity of the antibody population obtained toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However, because other plasma constituents contributed to a nonspecific basal signal in the RIA, a procedure by which the peptide could be reproducibly extracted from plasma was developed. The mean level of this species in normal individuals younger than the age of 40 was 66.4 pmol/L, and elevations up to 550 pmol/L were observed in patients with evidence of disseminated intravascular coagulation. The validity of these measurements of factor X activation is supported by the fact that the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native peptide and can be quantitatively recovered. The mean concentration of the activation fragment was markedly decreased to 25.7 pmol/L in patients with hereditary factor VII deficiency (P = .0001 v normal controls), whereas the mean level in subjects with factor VIII deficiency was 61.1 pmol/L (P greater than .1 v normal controls). These data indicate that the basal (ie, in the absence of thrombosis or provocative stimuli) levels of FXP under in vivo conditions result mainly from the activity of the extrinsic pathway.

Reports of dermatomyositis and polymyositis during pregnancy are rare, there being only one previously published case that we know of in the literature. We report a case in which severe polymyositis developed during pregnancy and the patient experienced dramatic improvement following delivery. Since there is evidence that polymyositis is an immune-mediated disease, one has to speculate that the fetus and its complement of foreign antigens might have played a role in the patient's disease.