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Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in vivo. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high affinity VEGF receptors. VEGF plays an essential role in developmental angiogenesis and is important also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with VEGF inhibitors in a variety of malignancies are ongoing. Recently, a humanized anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the FDA as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.

Background: Fibrin formation is required for tumor angiogenesis, metastasis, and invasion. D-dimer, a fibrin degradation product, is produced when crosslinked fibrin is degraded by plasmin. The current study prospectively examined D-dimer levels in patients with metastatic colorectal carcinoma treated in a Phase II randomized trial comparing bevacizumab (Avastin, Genentech, South San Francisco, CA) plus 5-fluorouracil/leucovorin (5-FU/LV) with 5-FU/LV alone., Methods: At least one circulating D-dimer level was evaluable in 98 of the 104 previously untreated patients with metastatic colorectal carcinoma in the current trial. Plasma D-dimer levels were determined using a quantitative immunoassay kit at enrollment, before each treatment, and at the time of trial completion or disease progression., Results: At trial enrollment, 86 of 104 patients (88%) had elevated D-dimer levels (> 20 ng/mL), and 86 of 102 patients (84%) had elevated carcinoembryonic antigen (CEA) levels (> 3 ng/mL). Baseline D-dimer levels were correlated with the following baseline characteristics: CEA (Pearson coefficient, 0.31; P = 0.002), albumin levels (Pearson coefficient, -0.32; P = 0.002), tumor burden (Pearson coefficient, 0.30; P = 0.003), and number of metastatic sites (Pearson coefficient, 0.21; P = 0.04). At the time of progression, plasma D-dimer levels reached a maximum postbaseline value in 51 of 61 patients (84%), whereas the CEA level was at its maximum postbaseline value in 39 of 55 patients (71%). Baseline D-dimer levels were a strong predictor of overall survival on univariate analysis (P = 0.008) and multivariate analysis (P = 0.03). Overall, treatment with bevacizumab (5 mg/kg) and baseline D-dimer levels were the only predictors of overall survival (P < 0.05)., Conclusions: The current study indicates that fibrin remodeling is an important prognostic feature in metastatic colorectal carcinoma. D-dimer levels should be incorporated into prognostic models, and D-dimer may represent a useful biomarker for patients treated with antiangiogenic agents., (Copyright 2004 American Cancer Society.)

Following confirmation of the appropriate dosage, safety and potential efficacy of Herceptin(trastuzumab) in small-scale phase I and II trials involving patients with refractory disease, a large trial was conducted in 222 patients with breast cancer who had relapsed after one or two chemotherapy regimens for their metastatic disease. The results showed a positive and durable overall response rate (15% according to a response evaluation committee (REC) assessment) using trastuzumab monotherapy (initial dose 4 mg/kg intravenously (i.v.) followed by 2 mg/kg i.v. weekly). In another recently completed phase II trial, 113 patients were randomised to two dose levels (initial dose of 4 mg/kg i.v. dose followed by 2 mg/kg i.v. weekly, or initial dose of 8 mg/kg followed by 4 mg/kg i.v. weekly) of single-agent trastuzumab as first-line therapy for metastatic disease. The preliminary overall response rate was 23% based on investigator assessment, and tolerability was excellent as in previous trials; efficacy was similar in both dose groups, but the side-effects tended to be more frequent in the higher dose group. The preferred dosage is therefore the same as that currently recommended, i.e. an initial dose of 4 mg/kg i.v. followed by 2 mg/kg weekly i.v. until disease progression.

The pivotal phase II and III Herceptin trials proved the efficacy and safety of second- or third-line single-agent Herceptin and first-line Herceptin in combination with chemotherapy, respectively. In the current trial, 114 patients were randomized to one of two dose groups of first-line Herceptin monotherapy: standard dose of 4 mg/ kg initial dose followed by 2 mg/kg intravenous (i.v.) weekly; or high dose of 8 mg/kg initial dose followed by 4 mg/kg i.v. weekly. The regimen was generally well tolerated. A similar incidence of adverse events was demonstrated in the two dose groups with the possible exception of acute infusion-related events such as fever and chills as well as rash and dyspnea, which appear to be more prevalent in the higher dose group. The overall response rate was 26% and response rates were similar between the two dose groups (24% for the standard Herceptin dose group and 28% for the high Herceptin dose group). Subgroup analysis determined a higher response rate in IHC 3+ patients (35%) and FISH-positive patients (41%). When women with stable disease for > or =6 months were included with responders, the clinical benefit rate in IHC 3+ patients was 47%. Median survival was 24.4 months, which is comparable with the survival rate seen in the pivotal phase III combination trial (25 months). Therefore, single-agent Herceptin is an important new option for the first-line treatment of HER2-positive metastatic breast cancer patients., (Copyright 2001 S. Karger AG, Basel)

Following confirmation of the appropriate dosage, safety and potential efficacy of Herceptin(R) (trastuzumab) in small-scale phase I and II trials involving patients with refractory disease, a large trial was conducted in 222 patients with breast cancer who had relapsed after one or two chemotherapy regimens for their metastatic disease. The results showed a positive and durable overall response rate (15% according to a response evaluation committee (REC) assessment) using trastuzumab monotherapy (initial dose 4 mg/kg intravenously (i.v.) followed by 2 mg/kg i.v. weekly). In another recently completed phase II trial, 113 patients were randomised to two dose levels (initial dose of 4 mg/kg i.v. dose followed by 2 mg/kg i.v. weekly, or initial dose of 8 mg/kg followed by 4 mg/kg i.v. weekly) of single-agent trastuzumab as first-line therapy for metastatic disease. The preliminary overall response rate was 23% based on investigator assessment, and tolerability was excellent as in previous trials; efficacy was similar in both dose groups, but the side-effects tended to be more frequent in the higher dose group. The preferred dosage is therefore the same as that currently recommended, i.e. an initial dose of 4 mg/kg i.v. followed by 2 mg/kg weekly i.v. until disease progression.

Sibrafiban is a double prodrug that is converted to the inactive single prodrug and to the active GP IIb/IIIa antagonist after oral administration. This clinical investigation evaluated whether coadministration of oral aspirin or intravenous heparin would alter the pharmacokinetics or pharmacodynamics of oral sibrafiban. Twenty-four adult subjects received two of the following four combinations: sibrafiban alone, sibrafiban with ASA, sibrafiban with heparin, and sibrafiban with ASA and heparin, separated by a 2-week washout period. Concentration profiles of active drug in citrate and EDTA plasma were unchanged with coadministration of ASA or heparin. No pharmacodynamic interaction was seen with coadministration of heparin. Inhibition of platelet aggregation increased 4% to 55%, and Ivy bleeding time increased 58% to 87% with coadministration of sibrafiban and ASA. The combined pharmacodynamic effect of sibrafiban and ASA may indicate a potentially greater therapeutic effect but an increased risk of bleeding when these drugs are used in combination.

Background: Exposure to indoor fungus growth and tobacco smoke has been epidemiologically linked to unexplained pulmonary hemorrhage in infants., Objective: To describe the 40-day-old male infant who had been exposed to fungi for a discrete 2-week period followed by acute exposure to environmental tobacco smoke prior to development of a life-threatening pulmonary hemorrhage., Patient and Methods: History and clinical evaluation of the infant immediately followed the pulmonary hemorrhage. Air and surface sampling for isolation and identification of fungal growth in the dwelling where the infant resided before the acute hemorrhage was accomplished when the homeowner returned from vacation 4 months after the clinical event., Results: Two fungi associated with mycotoxin production were cultured from surface samples collected in the residence: Penicillium (possibly Penicillium purpurogenum) and a Trichoderma species. Stachybotrys atra was not isolated from air or surface samples. Environmental tobacco smoke exposure occurred over a discrete several-hour period prior to onset of the acute pulmonary hemorrhage., Conclusions: Avoidance of unnecessary exposure of infants to fungus growth in water-damaged environments or exposure to tobacco smoke is prudent. Further investigation into the toxic effects of indoor fungi as causes of infantile pulmonary hemorrhage is warranted.

Sibrafiban is a double prodrug that is converted to the inactive single prodrug and to the active IIb/IIIa antagonist following oral administration. Pharmacokinetics (PK) and pharmacodynamics (PD) of oral sibrafiban and its metabolites were evaluated in patients postacute coronary syndrome receiving once- or twice-daily sibrafiban for up to 28 days at several dose levels. Mean peak concentrations of sibrafiban were < 5 ng/mL. Peak single prodrug concentrations occurred 1.7 +/- 1.0 (mean +/- SD) hours after sibrafiban dosing. Total apparent plasma clearance of the single prodrug was 40 +/- 15 L/h, and the elimination half-life was 2.3 +/- 0.8 hours. Mean values of the steady-state pharmacokinetics for total concentrations of the active drug over all doses were: time to peak plasma concentration, 5.0 +/- 1.7 hours; apparent clearance, 13.9 +/- 3.9 L/h; and half-life, 11.0 +/- 2.8 hours. Once-daily dosing resulted in high peak-trough excursions in active drug concentrations: trough concentrations were 21% +/- 6% of peak. Twice-daily dosing resulted in an AUC for the active drug on Day 28 that was 168% +/- 36% of that on Day 1, and steady-state trough concentrations were 54% +/- 10% of peak with sustained inhibition of platelet aggregation. Dose-adjusted steady-state active drug concentrations increased with increasing age and with decreasing renal function and body weight.

Unlabelled: This study was designed to determine the magnitude and time course of platelet activation during therapy of acute coronary syndromes with an oral platelet antagonist., Background: Platelet activation and aggregation are central to the pathogenesis of the acute coronary syndromes (ACS). However, few data are available on levels of platelet activation over time in patients with ACS, especially in the setting of chronic glycoprotein (GP) IIb/IIIa inhibition., Methods: The Thrombolysis in Myocardial Infarction (TIMI) 12 trial was a phase II, double-blind trial evaluating the effects of sibrafiban, an oral, selective antagonist of the platelet glycoprotein IIb/IIIa receptor in patients stabilized after an ACS. A subset of 90 of the 329 patients in the study had measurement of platelet activation as assessed by the expression of platelet associated P-Selectin on days 0, 7 and 28. Platelet activation was measured in blood samples that were fixed either immediately (spontaneous activation) or after 5 minute incubation with 0, 1 microM or 5 microM ADP in order to assess platelet responsiveness to very low or moderate stimulation., Results: At baseline there was a significant elevation of spontaneous platelet activation as compared to samples obtained from normal donors or from patients who did not have acute coronary syndromes (ACS patients 27.6+/-18.7%, Normal controls 8.5+/-4.4%, Patient controls 10.9+/-7.1%, p < 0.005 for both). In addition, there was a significant decrease in the levels of platelet activation with time during the 28 days of treatment with sibrafiban. Nevertheless, even on day 28, the TIMI-12 patients continued to show elevated platelet activation in comparison to the control groups (p < 0.05 for both)., Conclusions: These results suggest that platelets remain activated long after clinical stabilization post ACS. Although platelet activation decreased after one month of oral GPIIb/IIIa inhibition, levels remained higher than normal, suggesting the need for long-term antiplatelet therapy following ACS.

Objective: Prospective study of the efficacy of calf lung surfactant extract in pediatric respiratory failure., Design: Multi-institutional, prospective, randomized, controlled, unblinded trial., Setting: Eight pediatric intensive care units (ICU) of tertiary medical centers., Patients: Forty-two children with acute hypoxemic respiratory failure characterized by diffuse, bilateral pulmonary infiltrates, need for ventilatory support, and an oxygenation index of >7., Intervention: Instillation of intratracheal surfactant (80 mL/m2)., Measurements and Main Results: Ventilator parameters, arterial blood gases, and derived oxygenation and ventilation indices were recorded before and at intervals after surfactant administration. Complications and outcome measures, including mortality, duration of mechanical ventilation, and length of pediatric ICU and hospital stay, were also examined. Patients who received surfactant demonstrated rapid improvement in oxygenation and, on average, were extubated 4.2 days (32%) sooner and spent 5 fewer days (30%) in pediatric intensive care than control patients. There was no difference in mortality or overall hospital stay. Surfactant administration was associated with no serious adverse effects., Conclusions: Administration of calf lung surfactant extract, calfactant, appears to be safe and is associated with rapid improvement in oxygenation, earlier extubation, and decreased requirement for intensive care in children with acute hypoxemic respiratory failure. Further study is needed, however, before widespread use in pediatric respiratory failure can be recommended.

Background: Inhibitors of the platelet glycoprotein IIb/IIIa receptor given intravenously have been shown to be effective in reducing ischemic complications after coronary angioplasty and in unstable angina, making this a promising new class of agents for the treatment and prevention of ischemic events in patients with acute coronary syndromes. Sibrafiban (Ro 48-3657) is an oral, peptidomimetic, selective antagonist of the glycoprotein IIb/IIIa receptor., Methods and Results: The Thrombolysis in Myocardial Infarction (TIMI) 12 trial was a phase II, double-blind, dose-ranging trial designed to evaluate the pharmacokinetics (PK), pharmacodynamics (PD), safety, and tolerability of sibrafiban in 329 patients after acute coronary syndromes. In the PK/PD cohort of TIMI 12, 106 patients were randomized to receive one of seven dosing regimens of sibrafiban, ranging from 5 mg daily to 10 mg twice daily for 28 days. In the safety cohort, 223 patients were randomized to one of four dose regimens of sibrafiban (ranging from 5 mg twice daily to 15 mg once daily) or aspirin for 28 days. High levels of platelet inhibition were achieved: mean peak values ranged from 47% to 97% inhibition of 20 micromol/L ADP-induced platelet aggregation on day 28 across the seven doses. Twice-daily dosing provided more sustained platelet inhibition (mean inhibition, 36% to 86% on day 28), whereas platelet inhibition returned to baseline levels by 24 hours with once-daily dosing. Major hemorrhage occurred in 1.5% of patients treated with sibrafiban and in 1.9% of patients treated with aspirin. Protocol-defined "minor" bleeding, usually mucocutaneous, occurred in 0% to 32% of patients in the various sibrafiban groups and in none of the patients treated with aspirin. Minor bleeding was related to total daily dose (P=.002), once- versus twice-daily dosing (P<.0001), renal function (P<.0001), and presentation with unstable angina (P<.01)., Conclusions: The oral glycoprotein IIb/IIIa antagonist sibrafiban achieved effective, long-term platelet inhibition with a clear dose-response but at the expense of a relatively high incidence of minor bleeding. Oral IIb/IIIa inhibition deserves further study as a new treatment strategy in patients after acute coronary syndromes.

In the development of antisense therapeutics, there have been a number of hybridization-independent effects characterized for phosphorothioate oligodeoxynucleotides. One such effect is the transient prolongation of clotting times following intravenous infusion of high doses. In this study, inhibition of clotting times was characterized by determining the time course of both APTT and plasma oligonucleotide following intravenous infusion of ISIS 2302 in cynomolgus monkeys. Prolongation of APTT was also achieved by addition of ISIS 2302 to citrated blood from untreated monkeys, allowing the investigation of the mechanism of inhibition in vitro. Results from this study clearly indicate that the intrinsic pathway (APTT) was more sensitive to inhibition than the extrinsic pathway (PT). The prolongation of APTT was also shown to be transient and closely correlated with plasma oligonucleotide concentrations. The extent of APTT prolongation can be controlled by minimizing peak plasma oligonucleotide concentrations through lowering the dose or prolonging infusion duration. Direct addition of ISIS 2302 to blood produced quantitatively similar inhibition of clotting times. This effect was similar for a number of different phosphorothioate oligodeoxynucleotides, but oligonucleotides containing phosphodiester linkages and 2'-propoxy linkages were much less inhibitory. Additional in vitro studies indicated that the mechanism of inhibition was independent of that of heparin and possibly involved selective inhibition of the intrinsic pathway as well as the common clotting pathway. Investigation of selective clotting factors indicated that there was no direct inhibition of the enzymatic activity of factor Xa, XIa, or thrombin using chromogenic substrates. However, ISIS 2302 did produce a concentration-dependent increase in clotting time when fibrinogen was used as the substrate for thrombin.

Congenital hypoprothrombinemias are very rare, inherited disorders in which factor II (prothrombin) levels and/or activity are extremely low or absent. We report eight pregnancies in a patient with this disorder. Obstetric complications attributed to the coagulation disturbance included first-trimester bleeding in each pregnancy, miscarriage in four of the pregnancies, spontaneous maternal subarachnoid hemorrhage in one, and postpartum hemorrhage in one of four term pregnancies despite administration of clotting factor concentrate. The management of pregnancy in congenital hypoprothrombinemia, and issues of coagulation factor replacement, are discussed.

Diamine oxidase (histaminase), an enzyme that oxidatively deaminates putrescine and histamine, was purified from human placenta and from pig kidney. Both NH2-terminal sequences are highly homologous to the human kidney amiloride-binding protein, previously thought to be a component of the amiloride-sensitive Na+ channel. Monoclonal antibodies raised against the pig kidney amiloride-binding protein immunoprecipitate a polypeptide with the same M(r) (105,000) as that of pig kidney diamine oxidase. That polypeptide has both diamine oxidase activity and the capacity to bind [3H]phenamil, a tritiated amiloride derivative. Cells stably transfected with human kidney amiloride-binding protein cDNA express a high diamine oxidase activity. In transfected cells as well as with the purified enzyme, this activity was inhibited by amiloride and by some of its derivatives, such as phenamil and ethylpropylamiloride. Amiloride inhibition seems to be due to drug binding at the active site of the enzyme. These data indicate that human placental diamine oxidase is identical to the human kidney amiloride-binding protein and that amiloride analogues may have wider physiological effects besides those on epithelial ion transport.

The heparin-releasable proteins are a group of proteins that are targeted to the endothelial surface by attachment to glycosaminoglycans and may have functions specific to the endothelium-blood interface. In this study, heparin-affinity chromatography of human postheparin plasma was used as a method to identify and study novel heparin-releasable proteins. Six proteins seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels have increased levels in plasma after intravenous heparin. The six proteins are platelet factor 4, midkine, pleiotrophin, and several novel proteins. Midkine and pleiotrophin are related cytokines that are developmentally regulated, neurotrophic, and mitogenic. Additional studies show that levels of midkine and pleiotrophin peak at 10 to 30 minutes after injection of heparin. Heparin-releasable midkine and pleiotrophin do not originate from blood cells or the kidney. Heparin-releasable midkine may originate from endothelial cells. Soft agar culture of an adenocarcinoma cell line (SW-13) demonstrates growth-stimulating activity similar to that described for pleiotrophin in the heparin-agarose eluate of postheparin plasma but not in the heparin-agarose eluate of preheparin plasma. It is concluded there are more heparin-releasable proteins than previously identified, including midkine and pleiotrophin, and that heparin-affinity chromatography of postheparin plasma is a useful technique for identifying novel heparin-releasable proteins.

Eight cases of invasive group A streptococcal disease in young children were reported over a three-month period, February to April 1990. The spectrum of clinical disease included: pneumonia with bacteremia (two patients), osteomyelitis/septic arthritis (three patients), epiglottitis/supraglottitis (two patients), and sepsis without a focus (one patient). Three cases followed chicken pox. Three children were in shock at the time of presentation, including one child who had a toxic shock-like appearance. Only four children had pharyngitis. Bacteremia was confirmed in three children and presumed in another three. All the subjects survived. Four isolates of group A streptococci were tested for exotoxin A, B, and C (A-0, B-4, C-1) production. These data confirm the reappearance of a highly invasive strain of group A streptococci capable of producing a variety of clinical diseases, including bacteremia and shock, in a significant proportion of victims.

Fifteen patients undergoing major surgical procedures were evaluated for lipoprotein associated coagulation inhibitor (LACI) antigen, factor VII (F VII), antithrombin III (AT III), and peripheral blood monocyte tissue factor (TF) activity immediately before surgery and on following days. A peak in monocyte TF activity occurred between the first and fifth days after surgery in 10 of the patients, while LACI, F VII, and AT III levels dropped in a qualitatively parallel manner in 8 of these patients. LACI, F VII, and AT III levels decreased after surgery in two additional patients even though TF activity also decreased after surgery in these patients. In the remaining 3 patients who developed infections during the study, TF activity rose within 2 days of the diagnosis of infection in addition to the postoperative peak. In two of these patients, LACI levels increased dramatically near the end of the study period without concomitant changes in F VII and AT III. Overall, the presurgical TF levels in disrupted monocytes varied 52-fold and the maximal TF activity varied 24-fold among patients. The TF response following surgery is therefore heterogenous in both temporal occurrence and magnitude of the postsurgical peak. The patients also varied considerably in the presurgical levels of monocyte TF activity. A possible association between the level of presurgical TF activity and the magnitude of the postsurgical peak was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits the tissue factor/factor VIIa/calcium/phospholipid complex in a factor Xa-dependent manner. Recombinant LACI (rLACI) added exogenously to plasma prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) as a function of rLACI concentration in linear and curvilinear manners, respectively. Under these standard assay conditions, the amounts of rLACI required to double the APTT and PT were approximately 350- and 90-fold the plasma concentration of LACI, respectively. Likewise, addition of antibodies against LACI to pooled normal, factor VIII-deficient, or factor IX-deficient plasma had no effect on their respective APTTs and PTs, demonstrating the insensitivity of these assays to endogenous LACI. The prothrombin time assay was modified by using dilute thromboplastin. Unlike the standard prothrombin time assay, the clotting times were prolonged for factors VIII- or IX-deficient plasma relative to pooled normal plasma in this modified PT assay. Additionally, the degree of factor deficiency, as determined by the APTT assay, was correlated with that determined by the modified PT assay using dilute thromboplastin. When antibodies against LACI were added to pooled normal, factor VIII-deficient, or factor IX-deficient plasma and the prothrombin time assay initiated using dilute thromboplastin, the clotting times for antibody-treated plasma were shorter than for the corresponding plasma in the absence of antibodies. Moreover, the clotting times for factors VIII- and IX-deficient plasmas treated with antibodies raised against LACI were at least as fast as pooled normal plasma in the absence of LACI antibodies when dilute thromboplastin was used to initiate clotting. These results suggest that the prothrombin time assay using dilute thromboplastin may more accurately reflect what occurs in vivo and that LACI may play an important role in the prolonged bleeding of those with hemophilia A or B.

Human plasma contains an inhibitor of tissue factor-initiated coagulation known as the lipoprotein-associated coagulation inhibitor (LACI) or also known as the extrinsic pathway inhibitor (EPI). A competitive fluorescent immunoassay was developed to measure the plasma concentration of LACI in samples from normal individuals and patients with a variety of diseases. The LACI concentration in an adult control population varied from 60% to 160% of the mean with a mean value corresponding to 89 ng/mL or 2.25 nmol/L. Plasma LACI levels were not decreased in patients with severe chronic hepatic failure, warfarin therapy, primary pulmonary hypertension, thrombosis, or the lupus anticoagulant. Plasma LACI antigen was decreased in some, but not all patients with gram-negative bacteremia and evidence for disseminated intravascular coagulation. Plasma LACI levels were elevated in women undergoing the early stages of labor (29%), in patients receiving intravenous tissue-type plasminogen activator (45%), and in patients receiving intravenous heparin (375%). A radioligand blot of the pre- and post-heparin plasma samples shows the increase to be in a 40-Kd form of LACI. Very low levels of plasma LACI antigen were found in patients with homozygous abetalipoproteinemia and hypobetalipoproteinemia, diseases associated with low plasma levels of apolipoprotein B containing lipoproteins. Following the injection of heparin into one patient with homozygous abetalipoproteinemia, the plasma LACI antigen level increased to a level comparable with that in normal individuals after heparin treatment.

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin-releasable LACI (HRL), as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

TF mediated initiation of coagulation appears to play a critical role in normal hemostasis and probably pathologic thrombosis as well. Although teleological considerations would seem to suggest that a specific regulator of this process should exist, and although the presence in plasma of such an inhibitor was documented many years ago, it was not until the past five years that the inhibitor was characterized and its mechanism of action defined. LACI produces factor Xa-dependent feedback initiation of the VIIa/TF catalytic complex. The mechanism of this feedback inhibition is novel. First, LACI, a multi-headed protease inhibitor, binds factor Xa, a product of VIIa/TF catalysis, at one of its inhibitory domains. The Xa-LACI complex, possibly acting as a pseudosubstrate, then is able to bind to VIIa/TF in an appropriate conformation such that a second inhibitory domain of LACI is positioned to interact with factor VIIa in the VIIa/TF complex. Whether such a unique means of eliciting feedback inhibition in a protease cascade is repeated in nature is unknown. The existence of LACI appears to help explain the clinical need for both "extrinsic" and "intrinsic" coagulation pathways. In addition, data to the present are consistent with the notion that, in normal hemostasis at least, TF is responsible for an initial burst of factor Xa generation which provides sufficient thrombin to induce the aggregation of platelets and the activation of the critical coagulation cofactors factor V and factor VIII. Ultimate and persistent hemostasis, however, appears to require the continued production of additional factor Xa through the action of factor IXa and factor VIII. The fact that patients with factor XI deficiency suffers a variable but usually mild bleeding diathesis suggests that under certain conditions the initial burst of factor IXa formed through the action of VIIa/TF is insufficient and supplemental factor IXa generated by factor XIa is needed for normal hemostasis. The mechanism by which this factor XIa is generated in vivo, however, has not been determined. We stress that the predicted in vivo role of LACI is simply that--a prediction based on its known in vitro properties. Documentation of its physiologic importance remains to be provided and is an area of active research. Further, although significant progress has been made over the past few years in the characterization of LACI, many questions remain unanswered. For example: What is the mechanism for LACI's association with lipoproteins in plasma? What function, if any, does the third Kunitz-type protease inhibitor domain in LACI serve? (ABSTRACT TRUNCATED AT 400 WORDS)

Lipoprotein-associated coagulation inhibitor (LACI) appears to inhibit tissue factor (TF)-induced blood coagulation by forming a quaternary inhibitory complex containing factor Xa, LACI, factor VIIa, and TF. A genetically engineered hybrid protein consisting of the light chain of factor Xa and the first Kunitz-type inhibitor domain of LACI is shown to directly inhibit the activity of the factor VIIa-TF catalytic complex. Unlike inhibition of factor VIIa-TF activity by native LACI, inhibition by the hybrid protein is not dependent on factor Xa. In an assay of TF-induced coagulation, 50% TF inhibition occurs with hybrid protein at 35 nanograms per milliliter, whereas LACI at 2.5 micrograms per milliliter is required for an equivalent effect. gamma-Carboxylation of glutamic acid residues in the factor Xa light chain portion of the hybrid protein is required for inhibitory activity, indicating that the first Kunitz-type domain of LACI alone is not sufficient for inhibition of factor VIIa-TF.

Lipoprotein-Associated Coagulation Inhibitor (LACI) is a factor Xa dependent inhibitor of the factor VII(a)/Tissue Factor catalytic complex. Deduced from partial cDNA sequence, LACI's amino acid sequence has recently been reported. Northern blot analysis showed LACI cDNA hybridizes to RNAs of 1.4 and 4.0 kb in size. To complete the characterization of the LACI message(s), overlapping LACI cDNAs were isolated from a human endothelial cell library. Sequence analysis revealed the clones' inserts span 4023 bases of sequence, consisting of 381 bases of 5' untranslated sequence, an open reading frame of 912 bases, 2682 bases of 3' untranslated sequence and 48 bases of poly(A) sequence. In addition, a short 1.4 kb insert which encodes for LACI was found to contain 49 bases of 3' untranslated sequence and a 3' poly(A) tail. The 1.4 kb of sequence is contained in the 4.0 kb sequence, except for 14 bases of 5' sequence, suggesting that the LACI messages arise by the use of alternative termination and polyadenylation signals during processing. Northern blot analysis of RNA isolated from cells treated with actinomycin D showed both RNA species appear to be relatively stable. Using a bovine papilloma virus vector, LACI cDNA was transfected into mouse C127 fibroblasts. The recombinant LACI is recognized by polyclonal anti-LACI IgG, binds to factor Xa and inhibits VII(a)/Tissue Factor activity in a similar fashion as LACI purified from HepG2 cell conditioned media.

Stimulation with thrombin or the calcium ionophore, A23187 caused human platelets to release a coagulation inhibitor similar to the Lipoprotein Associated Coagulation Inhibitor (LACI). This was documented functionally, with clotting assays measuring tissue factor inhibition and factor Xa inhibition, as well as immunologically, in a competitive immunoassay. The total amount of LACI released by 3 x 10(8) platelets after two hours stimulation was 7% to 8% of the amount found in 1 mL of serum. Half of the LACI was released by five minutes. The LACI was present in the platelet supernatant and was not associated with the platelet membrane or shed vesicles. The tissue factor and factor Xa inhibitory activities that were released were neutralized by preincubating the platelet supernatants with specific rabbit polyclonal anti-LACI IgG. On Western blot, platelet LACI appeared to run as a doublet with a molecular weight (mol wt) 45,000 to 47,000. Blood samples obtained from the site of a wound (template bleeding time) demonstrated a progressive increase in LACI concentration. A cDNA probe, derived from endothelial cell LACI cDNA, hybridized selectively to 4.0 and 1.4 kb transcripts in a preparation of platelet mRNA.

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood coagulation can be initiated when factor VII or VIIa, a plasma protease, binds to its essential cofactor, tissue factor (TF), and proteolytically activates factors IX and X, triggering a cascade of events which eventually leads to the formation of thrombin and a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits activated factor X (Xa) directly and, in a Xa-dependent way, inhibits VII(a)/TF activity, presumably by forming a quaternary Xa/LACI/VII(a)/TF complex. Sequence analysis of complementary DNA clones has shown that LACI contains three tandemly repeated Kunitz-type serine protease inhibitory domains. To investigate the relationship between these Kunitz structures and LACI function, we have used site-directed mutagenesis to produce altered forms of LACI in which the residue at the active-site cleft of each Kunitz domain has been individually changed. The second Kunitz domain is required for efficient binding and inhibition of Xa, and both Kunitz domains 1 and 2 are required for the inhibition of VIIa/TF activity; but alteration of the active-site residue of the third Kunitz domain has no significant effect on either function. We propose that in the putative inhibitory complex, Kunitz domain 1 is bound to the active site of VII(a)/TF and that Kunitz domain 2 is bound to Xa's active site.