Characterization of the catalytic subunit of factor XIII by radioimmunoassay

Plasma factor XIII is composed of two subunits, a and b, whereas platelet and other intracellular zymogens have only a-subunits. The catalytic subunit, a, is the same in all forms. In order to characterize the interactions of 1- with b-chains in the plasma zymogen and a-chains with other molecules and to correlate factor XIII activity with a-protein, a specific, sensitive radioimmunoassay was developed. With the polyclonal antisera used, the assay recognizes all molecular forms of a (zymogens, activation intermediates, enzyme) equally well. The assay can be used to determine a-chain concentration in plasma and serum and in purified test systems. Fibrinogen in high concentrations affects the assay, probably by interfering with the interactions of 125I-a with antibody. However, at the plasma dilutions used in the assay, there is no significant fibrinogen effect. With this assay, the a-chain concentration in normal plasma is approximately 15 micrograms/ml. This compares with 14 micrograms/ml b-chain in plasma and indicates that all of the a- and b-chains in plasma probably circulate in the form of an equimolar zymogen complex. The serum concentration of a-protein is about 6% of the plasma concentration. There is a high correlation between a-protein and factor XIII activity.     

Severe bleeding complications caused by an autoantibody against the B subunit of plasma factor XIII: a novel form of acquired factor XIII deficiency

Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused life-threatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A(2)B(2) complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patient's recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%.     

Heparin-induced thrombocytopenia: studies with a new low molecular weight heparinoid, Org 10172

Studies were performed to determine the cross-reaction rate of the heparin-dependent antibody with Org 10172, a new low molecular weight heparinoid, and to investigate the effect of Org 10172 on platelet activation induced by the antibody. The plasmas of 17 patients with thrombocytopenia induced by standard heparin were shown, by platelet aggregation studies, to contain the heparin-dependent antibody. Of these 17 patient plasmas, only three cross-reacted with the heparinoid, producing a cross-reaction rate of 18%. When Org 10172 was added to a reaction mixture containing normal platelet-rich plasma, patient plasma, and standard heparin with non-cross-reacting plasmas, it inhibited platelet aggregation and thromboxane B2 production induced by the antibody, provided that the ratio of Org 10172 concentration (anti- Xa U/mL) to standard heparin concentration (IU/mL) exceeded 2.5 to 5.0. This inhibitory effect was observed only with platelet activation mediated by the antibody, but not by collagen (2 micrograms/mL) or ADP (5.0 mumol/L). Additionally, three of 17 patients with serious thrombosis, whose plasma showed no cross-reaction with the heparinoid, received Org 10172 treatment with a good response in each case. These findings suggest that Org 10172 may be a useful drug for the treatment of heparin-induced thrombocytopenia.     

Free protein S levels are elevated in familial C4b-binding protein deficiency

In plasma, 40% of the protein S is free and functions as a cofactor for the anticoagulant effects of activated protein C. The remaining 60% of protein S is complexed to C4b-binding protein and is functionally inactive. A family with hereditary C4b binding protein deficiency has been identified with C4b-binding protein levels in an affected father and daughter of 37 micrograms/mL and 23 micrograms/mL, respectively; these values are significantly below the normal range for this protein of 180 micrograms/mL +/- 44 micrograms/mL (mean +/- 2 SD). The total protein S (free + bound) is normal in these individuals (23.2 micrograms/mL and 17.8 micrograms/mL, respectively; normal 19.1 micrograms/mL +/- 6.0 micrograms/mL). The free protein S levels are markedly increased at 22.5 micrograms/mL and 17.4 micrograms/mL, respectively (normal 5.9 micrograms/mL +/- 2.4 micrograms/mL). This experiment of nature shows that total protein S levels in plasma are not affected by the absence of C4b-binding protein and that chronic elevation of free protein S is not associated with increased hemorrhagic tendencies.     

Biosynthesis of factor XIII B subunit by human hepatoma cell lines

The plasma transglutaminase, factor XIIIa (FXIIIa), circulates as a zymogen containing two proteins, A and B, arranged in a noncovalent tetrameric complex, A2B2. Biosynthesis of plasma FXIII has not previously been demonstrated. In the present study, direct evidence has been obtained that two human hepatoma cell lines, Hep G2 and PLC/PRF/5, synthesize and secrete FXIII B protein. Secretion of the B subunit of FXIII by Hep G2 was demonstrated by immunoblotting. De novo synthesis by Hep G2 was confirmed in 35S-methionine-labeled cultures. Radiolabeled conditioned medium was concentrated, mixed (1:1) with purified B protein, and examined by crossed immunoelectrophoresis with antiserum to the B subunit. The single protein precipitin arc of purified B protein comigrated with the radiolabeled FXIII from Hep G2 visualized by autoradiography, indicating both electrophoretic and antigenic identity. The data presented here represent the first demonstrations of biosynthesis of FXIII B protein by any cell type and suggest that the liver is the site of synthesis of FXIII B protein. Further analysis of concentrated Hep G2 serum-free conditioned medium (SFCM) and cell lysate by immunoblotting following nondenaturing agarose gel electrophoresis demonstrated the FXIII A protein as well as the B protein and also revealed synthesis and secretion of the A and B proteins by PLC/PRF/5. Crossed immunoelectrophoresis studies of Hep G2 SFCM and cell lysate suggest that Hep G2 cells also synthesize and secrete the plasma FXIII zymogen. With a specific radioimmunoassay for B protein, FXIII was found in Hep G2 SFCM at approximately 4 ng/mL; with an amplified rocket immunoelectrophoresis technique the level was approximately 5 ng/mL.     

Clot lysis induced by a monoclonal antibody against alpha 2-plasmin inhibitor

A monoclonal antibody (MoAb) to alpha 2-plasmin inhibitor designated JTPI-1 inhibited antiplasmin activity by interfering with formation of alpha 2-plasmin inhibitor (alpha 2-PI)-plasmin complex. With this MoAb, we observed plasma clot lysis in vitro and evaluated the potential of JTPI-1 to serve as a new therapeutic agent for thrombolysis. After adding 125I-labeled fibrinogen to plasma, clots were made by adding thrombin and calcium and were then resuspended in normal plasma containing various concentrations of JTPI-1. The presence of JTPI-1 enhanced release of the soluble 125I-labeled fibrin degradation fragment from the clots in a dose-dependent manner. With tissue plasminogen activator (t-PA)-depleted plasma, we showed that induction of clot lysis by JTPI-1 was dependent on fibrin-bound endogenous t-PA. Regulation of fibrinolysis initiated on the fibrin surface by fibrin-bound t-PA and plasminogen is mediated by alpha 2-PI cross-linked to fibrin by activated factor XIII. JTPI-1 bound to this cross-linked alpha 2-PI neutralized its activity and induced partial digestion of fibrin by plasmin. This resulted in additional binding of Glu-plasminogen to fibrin during the incubation. When 1.2 mumol/L JTPI-1 and 5 U/mL exogenous t-PA were present in the suspending plasma, the rate of clot lysis was essentially the same as that induced by 60 U/mL exogenous t-PA alone. These results suggest that JTPI-1 may be useful in reducing the amount of t-PA administered for thrombolytic therapy.     

Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased resistance to plasmic degradation of fibrin with highly crosslinked alpha-polymer chains formed at high factor XIII concentrations

We have previously demonstrated that increasing factor XIII concentrations above that present in plasma (1 U/mL) results in the formation of very high molecular weight alpha fate polyacrylamide and agarose gel electrophoresis (SDS-PAGE). In this report, we have examined the effect of such crosslinking on plasmic susceptibility of fibrin prepared from purified fibrinogen and from plasma in the presence of factor XIII concentrations between 0 and 10 U/mL. The crosslinking achieved with purified fibrinogen at 1 U/mL factor XIII increased resistance to plasmic degradation by 32% as measured in a radiolabeled clot lysis system. However, further increases in plasmic resistance occurred at factor XIII concentrations of 2 and 10 U/mL, the latter decreasing the lysis rate to 45% of that which occurred in the absence of factor XIII. To achieve the same rate of clot lysis with fibrin formed using 10 U/mL rather than 1 U/mL of factor XIII, an increase in plasmin concentration of up to 4.2-fold was required. Similar results were obtained using clots prepared from plasma in the presence of factor XIII concentrations greater than 1 U/mL. Since the alpha 2-plasmin inhibitor content was the same for fibrin at 1 or 10 U/mL factor XIII, the increasing plasmic resistance could not be attributed to increased binding of the inhibitor. We conclude that fibrin prepared in the presence of factor XIII at concentrations exceeding that in plasma shows increased resistance to plasmic degradation, which is likely explained by the formation of very high molecular weight alpha polymer chains.     

A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma.

A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA-12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu-PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.     

Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.     

Metabolic clearance rate (MCR) of dehydroepiandrosterone sulfate (DS), its metabolism to dehydroepiandrosterone, androstenedione, testosterone, and dihydrotestosterone, and the effect of increased plasma DS concentration on DS MCR in normal women

The combined infusion of [3H]dehydroepiandrosterone sulfate [( 3H]DS) and [14C]estrone (E1) for 6 h in four normal women in the follicular phase of the cycle demonstrated values of 13.77 +/- 0.27 L/day (mean +/- SE) for the DS MCR (MCRDS) and 1343 +/- 206 L/day for MCRE1. The fraction of DS metabolized to dehydroepiandrosterone (D), [rho]DS-D, was 0.285 +/- 0.044, and the fraction of D arising from DS (delta D) was 0.318 +/- 0.096, determined using isotopic techniques. Raising the plasma DS concentration by 8.6 micrograms/mL (23 mumol/L) by infusing DS iv increased the MCRDS to 24.49 L/day in the first subject, and raising the plasma DS by 11.40 +/- 0.33 micrograms/mL (30.94 +/- 0.90 mumol/L) in the next three subjects raised the MCRDS to 35.7 +/- 1.3 L/day (P less than 0.01) without changing the MCRE1. The increase in MCRDS produced by increasing plasma DS makes it difficult to accurately measure the MCRDS in effect at the subject's endogenous plasma DS concentration by infusion of nonisotopic DS. Determination of the fractions of D, androstenedione (delta delta 4A), and testosterone (T) arising from DS using the increase in the plasma concentrations of these steroids produced by infusion of nonisotopic DS gave the following estimates: delta D = 0.262 +/- 0.042, delta delta 4A = 0.089 +/- 0.046, and delta T = 0.273 +/- 0.14.     

Altered therapeutic range for quinidine after myocardial infarction and cardiac surgery

Although most assays for measuring drug levels in serum determine the total concentration, effects from a drug are determined better by measuring the concentration of unbound (free) drug in serum. When the free fraction of a drug is constant, the total drug concentration may act as a good guide in predicting drug activity. However, if the free fraction is altered from normal, the serum concentration of the total drug may be misinterpreted. Quinidine has a high binding affinity for alpha-1-acid glycoprotein. We present the case of a woman who had a myocardial infarction; after cardiac surgery, she was found to have high concentrations of alpha-1-acid glycoprotein (228 mg/dL) and a low free fraction of quinidine (0.032). At that time the patient had a total concentration of quinidine of 33.9 mumol/L (11 micrograms/mL) but showed no signs or symptoms of toxicity because the concentration of free quinidine was not high. Physicians should be aware of the limitations of assays in determining the unbound concentration of drugs in serum. This awareness is particularly crucial with drugs that bind well to serum proteins, especially when pathologic conditions change the extent of binding.     

Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects

Defects in platelet cytoplasmic Ca++ mobilization have been postulated but not well demonstrated in patients with inherited platelet secretion defects. We describe studies in a 42-year-old white woman, referred for evaluation of easy bruising, and her 23-year-old son. In both subjects, aggregation and 14C-serotonin secretion responses in platelet-rich plasma (PRP) to adenosine diphosphate (ADP), epinephrine, platelet activating factor (PAF), arachidonic acid (AA), U46619, and ionophore A23187 were markedly impaired. Platelet ADP and adenosine triphosphate (ATP), contents and thromboxane synthesis induced by thrombin and AA were normal. In quin2-loaded platelets, the basal intracellular Ca++ concentration, [Ca++]i, was normal; however, peak [Ca++]i measured in the presence of 1 mmol/L external Ca++ was consistently diminished following activation with ADP (25 mumol/L), PAF (20 mumol/L), collagen (5 micrograms/mL), U46619 (1 mumol/L), and thrombin (0.05 to 0.5 U/mL). In aequorin-loaded platelets, the peak [Ca++]i studied following thrombin (0.05 and 0.5 U/mL) stimulation was diminished. Myosin light chain phosphorylation following thrombin (0.05 to 0.5 U/mL) stimulation was comparable with that in the normal controls, while with ADP (25 mumol/L) it was more strikingly impaired in the propositus. We provide direct evidence that at least in some patients with inherited platelet secretion defects, agonist-induced Ca++ mobilization is impaired. This may be related to defects in phospholipase C activation. These patients provide a unique opportunity to obtain new insights into Ca++ mobilization in platelets.     

Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.     

A familial factor XIII subunit B deficiency

A 32-year-old woman with a bleeding tendency born of a consanguineous marriage, was found to have factor XIII subunit B deficiency. An abnormally low level of factor XIII activity was initially noticed and this finding led to further studies of the proband and her family. The notable features were: undetectable subunit B of factor XIII in the proband and her brother and reduced levels of subunit B, 34-52%, in her parents and children. The proband's brother had a markedly decreased level of subunit A protein. The level of factor XIII subunit A in platelets of the proband was normal. The half-life of subunit A determined from the disappearance curve of infused factor XIII subunit A concentrate was approximately 3 d and this is the shortest estimate of the half-life of factor XIII to date. From these results, it is suggested that subunit A is unstable in plasma deficient in subunit B and subunit B stabilizes the A protein. This is the first report of congenital deficiency of factor XIII subunit B and this disorder is thought to be inherited as an autosomal recessive trait.     

An essential role for lysophosphatidylcholine in the inhibition of platelet aggregation by secretory phospholipase A2

The release of secretory phospholipase A2 (sPLA2) into the mammalian circulation may contribute to the development of hemorrhagic and inflammatory diseases. sPLA2 has previously been shown to alter the behavior of platelets, leukocytes, and endothelial cells, although the molecular basis for these cellular effects has not been established. Our studies indicate that the inhibition of platelet aggregation by snake, bee venom, and pancreatic sPLA2 is dependent on a plasma cofactor. This cofactor resides within the lipoprotein fraction of plasma, with 54%, 31%, and 11% of the activity present in the high- density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions, respectively. Delipidation of HDL and LDL was associated with the complete loss of platelet-inhibitory activity. Incubation of purified sPLA2 with the HDL fraction of plasma resulted in the time-dependent generation of lysophosphatidylcholine (lysoPC). The formation of lysoPC correlated with the inhibition of platelet aggregation. Purified lysoPC (10 to 100 micrograms/mL) inhibited platelet aggregation and dense granule release induced by thrombin (0.05 U/mL), collagen (1 micrograms/mL), ionophore A23187 (2 mumol/L), ADP (12.5 mumol/L), and adrenaline (3.2 mumol/L). The inhibition of platelet aggregation by lysoPC was dose-dependent and correlated with decreased fibrinogen binding to glycoprotein IIb-IIIa. Our studies indicate that the enzymatic generation of lysoPC from plasma lipoproteins is essential for the sPLA2-mediated inhibition of platelet activation in the presence of albumin. These results raise the possibility that the toxic effects of circulating sPLA2 may be due in part to the generation of the bioactive lysophospholipid, lysoPC.     

Elevation of plasma truncated elastase alpha 1-proteinase inhibitor complexes in patients with inflammatory lung diseases.

Human neutrophil elastase plays an important role in the development of several inflammatory lung diseases; however, there have been relatively few investigations using plasma samples. In this report, we describe alterations in the plasma elastase:alpha 1-PI complex in patients with chronic obstructive pulmonary disease (COPD) (15 cases), COPD with infection (8), diffuse panbronchiolitis (DPB) (8), bronchiectasis (9), pneumonia (10), and the adult respiratory distress syndrome (ARDS) (14), and in 15 normal volunteers. The elastase:alpha 1-PI complex concentration was determined by an enzyme-linked immunosorbent assay. Western immunoblot analysis of the elastase:alpha 1-PI complex was also performed. Plasma elastase:alpha 1-PI complex was also performed. Plasma elastase:alpha 1-PI complex levels in patients with COPD with infection (504 micrograms/L +/- 93 micrograms/L) were significantly higher, as compared with those with COPD but without infection (118 micrograms/L +/- 9 micrograms/L) and normal volunteers (122 micrograms/L +/- 4 micrograms/L). Increased complex concentrations were also found in patients with DPB and bronchiectasis (643 micrograms/L +/- 222 micrograms/L and 558 micrograms/L +/- 198 micrograms/L, respectively) as compared with normal volunteers. Increased complex concentrations were also found in patients with pneumonia and ARDS (450 micrograms/L +/- 101 micrograms/L and 1,400 micrograms/L +/- 438 micrograms/L, respectively). Western immunoblot analysis using anti-alpha 1-PI antibody and antineutrophil elastase antibody showed two types of elastase:alpha 1-PI complexes, one with a molecular weight of 60,000 daltons (60 kilodaltons [KD]) and the other at 50,000 daltons (50 KD). Although the native 80-KD elastase:alpha 1-PI complex was detected in bronchoalveolar lavage fluid, it was not found in plasma. In summary, these results demonstrated that levels of the truncated complex were increased in patients with various inflammatory lung diseases. This truncated form may play an important role in the pathophysiology of inflammatory processes.     

A unique factor XIII inhibitor to a fibrin-binding site on factor XIIIA.

An 81-year-old woman, who presented with sudden episodes of spontaneous bleeding, was found to have a specific inhibitor of factor XIII. Her fibrin clots had approximately 70% gamma-gamma and no alpha polymer formation, under conditions where normal fibrin was fully cross-linked; the patient's clots were soluble in urea or monochloroacetic acid. Factor XIII activity in her plasma was 24%, measured by the dansylcadaverine incorporation assay. When mixed with normal plasma, the patient's plasma inhibited fibrin cross-linking; however, in mixtures of patient and normal plasma, there was no inhibition of factor XIII activity when assayed by the incorporation of dansylcadaverine into casein. Thus, this inhibitor was active against fibrin cross-linking but not against ligation of small molecules to casein. Consequently, gel electrophoresis of reduced, sodium dodecyl sulfate-solubilized fibrin clots was a simple, quantitative method that was used to measure inhibitor activity. This inhibitor is unique and has been designated inhibitor New Haven. It was neutralized by anti-IgG and anti-kappa. It did not inhibit the activation of factor XIII but did inhibit fibrin cross-linking. There was complex formation between the inhibitor and activated factor XIII (A', A*) but not between A2 or fibrinogen. Only A', A* and the 56-Kd fragment bound to affinity columns made with this IgG. The inhibitor significantly decreased the binding of A', A* to fibrin clots. These data indicate that the epitope for this inhibitor is in a fibrin binding site. It is hidden in the zymogen and expressed on A' and A*, indicating that the conformational change occurring with the cleavage of the activation peptide is sufficient to expose the fibrin binding site.     

Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.     

Molecular characterization of commercial porcine factor VIII concentrate

Commercial porcine factor VIII concentrate (Hyate:C) is effective in treatment of patients with hemophilia A who have circulating antibodies to factor VIII. The molecular forms of factor VIII in the concentrate were identified and evaluated in light of the known properties of porcine and human factor VIII. The factor VIII in the concentrate was isolated by tandem chromatography on gelatin-Sepharose and monoclonal anti-factor VIII-Sepharose. The factor VIII was 1% of the protein mass of the concentrate when calculated by either quantity of protein recovered or by radioimmunoassay. Both functional assay and Western blotting of the crude concentrate indicated that maximum coagulant function was achieved by proteolytic activation of procofactor forms of factor VIII. The factor VIII can be fractionated by cation-exchange high-performance liquid chromatography (HPLC) into two or three species of heterodimers depending on the lot. The specific activity of the purified porcine factor VIII was 550 U/mg using pooled porcine plasma at 1 U/mL as a standard. From this value, a factor VIII concentration in normal pig plasma of 2 micrograms/mL was calculated. This agreed well with a value of 3 micrograms/mL obtained by radioimmunoassay (RIA) of factor VIII in porcine plasma. In contrast, reported values for human factor VIII average 5800 U/mg, resulting in a calculated concentration in plasma of 0.2 microgram/mL. The finding that porcine plasma contains a significantly higher circulating mass of factor VIII than human plasma appears to explain previous difficulties in comparing porcine and human factor VIII in standard assays.