Prevention of venous thromboembolism after knee arthroscopy with low-molecular weight heparin (reviparin): Results of a randomized controlled trial.

PURPOSE: Deep venous thrombosis (DVT) is a common, important complication of major orthopaedic surgery, particularly knee arthroplasty. Knee arthroscopy is increasingly performed on an outpatient basis. Few reports have elucidated the incidence of venous thromboembolism (VTE) in patients undergoing arthroscopic surgery receiving no prophylaxis. The objective of the present trial was to evaluate the risk of VTE in those patients and to determine efficacy and safety of a low-molecular weight heparin (LMWH) in preventing VTE. TYPE OF STUDY: This is the first controlled randomized trial using objective diagnostic methods with blinded outcome assessment to reveal the incidence of VTE in outpatient arthroscopy and determine efficacy and safety of a LMWH (reviparin sodium) in preventing VTE in these patients. METHODS: There were 262 patients undergoing elective knee arthroscopy prospectively randomized to receive either no treatment or reviparin once daily subcutaneously for 7 to 10 days. The blindly assessed primary outcome measure was the incidence of DVT detected by compression color-coded sonography. Both groups were comparable with regard to demographics and baseline characteristics. RESULTS: 239 patients were evaluable (122 no treatment, 117 receiving LMWH). 6 DVT were detected - 5 in the control group (5/117 - 4.1%) and only one in the active treatment group (1/116 - 0.85%). This particular patient had a low level of protein C and a subnormal level of protein S. The odds ratio of 4.95 approximates a relative risk reduction of about 80%. Treatment with reviparin was safe and well tolerated. There was no major bleeding, four patients with minor bleedings. One patient had a transitory fall in platelet count below 100 giga-particles/L without any clinical symptoms. CONCLUSIONS: Patients undergoing knee arthroscopy have a moderate risk of VTE and effective prophylaxis can be achieved with LMWH (reviparin).

Evaluation of a sensitive colorimetric FXIII incorporation assay. Effects of FXIII Val34Leu, plasma fibrinogen concentration and congenital FXIII deficiency.

There is an increasing interest in the role of coagulation factor XIII (FXIII) in cardio- and cerebrovascular diseases. It has recently been reported that a common G-->T point mutation in the A-subunit gene of FXIII, which codes for a valine (val) to leucine (leu) change (FXIIIVal34Leu), is protective against thrombotic diseases but seems to increase the risk of intracerebral bleeding. We developed a colorimetric incorporation assay for detection of FXIII activity based on incorporation of 5-(biotinamido) pentylamine (BAPA) into fibrin or fibrinogen. With this new assay, we studied the effects of FXIIIVal34Leu mutation, plasma fibrinogen concentration and congenital FXIII deficiency on FXIII activity. There are no data available about the ability of different FXIII assays to detect altered activity in FXIIIVal34Leu genotypes. We therefore compared our results determined by the incorporation method with a commonly used photometric method based on ammonia release after cross-linking of glycine-ethylester to a specific glutamine containing peptide substrate. We also determined FXIII A-subunit antigen (Ag) levels using enzyme-linked immunosorbent assay (ELISA) technique. The FXIIIVal34Leu genotype could not be detected either by the photometric method nor by the FXIII A-subunit ELISA. The incorporation assay showed an increased specific FXIII activity in subjects possessing the leu allele. The photometric assay and ELISA gave similar results independent from genotype. In patients with congenital FXIII deficiency before and after substitution, however, ELISA and the incorporation assay gave similar results, whereas the photometric assay showed consistently higher values. Our results show that the incorporation assay, not the photometric assay based on ammonia release, can be used for detection of elevated activity in subjects with FXIIIVal34Leu. Because of specificity and over a wide range sensitivity, the assay can also be used for determination of FXIII deficiency and monitoring of FXIII substitution therapy.

Low levels of fibrin-stabilizing factor (factor XIII) in human Plasmodium falciparum malaria: correlation with clinical severity.

Plasmodium falciparum malaria is associated with procoagulant activity but not with thromboembolism. We measured coagulation factor XIII, i.e., fibrin-stabilizing factor, in 45 patients with falciparum malaria over time. Of these, 22 had organ complications. The factor XIII antigen (subunits A and B) and plasma activity levels were abnormally low in those with falciparum malaria. They increased during antiparasitic therapy. In 14 of 22 patients with complications, but in no patient with mild disease (P < 0.001), subunit A and activity was < 50%. The factor X.III levels were inversely correlated with clinical severity, parasitemia, and human neutrophil elastase (HNE), but not with thrombin-antithrombin III levels. Thus, low factor XIII levels may reflect proteolysis by HNE, rather than procoagulant activity. One could speculate that factor XIII degradation in severe malaria prevents thromboembolism. On the other hand, factor XIII deficiency might reduce protection of the vascular endothelium against HNE and reactive oxygen species, which would promote organ damage.

Factor XIIIa cross-linking of the Marburg fibrin: formation of alpham.gamman-heteromultimers and the alpha-chain-linked albumin. gamma complex, and disturbed protofibril assembly resulting in acquisition of plasmin resistance relevant to thrombophila.

The truncated Aalpha-chain of fibrinogen Marburg is partly linked with albumin by a disulfide bond. Based on the recovery of the first six amino acid residues assigned to the subunit polypeptides of fibrinogen (the Aalpha-and gamma-chains) and albumin, 0.33 mol of albumin was estimated to be linked to 1 mol of the Marburg fibrinogen. When the Marburg fibrinogen was clotted with thrombin-factor XIIIa-Ca2+, various alpham gamman heteromultimers were produced, and part of the albumin was cross-linked to the gamma-chain. Acid-solubilized Marburg fibrin monomer failed to form large aggregates that could be detected by monitoring turbidity at A350, but it was able to enhance tissue-type plasminogen-activator-catalyzed plasmin generation, though not as avidly as the normal control, indicating that the double-stranded protofibrils had, to some extent, been constructed. This idea seems to be supported by normal factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains. However, the cross-linked Marburg fibrin, being apparently fragile and translucent, was highly resistant against plasmin, and its subunit components were considerably retained for 48 hours as noted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the exact mechanisms are still unclear, the albumin-incorporated factor XIIIa-cross-linked Marburg fibrin seems to have undergone a critical structural alteration(s) to acquire resistance against plasmin. This aquisition of plasmin resistance may be contributed to the postoperative pelvic vein thrombosis and recurrent pulmonary embolisms in the patient after caesarian section for her first delivery at the age of 20 years.

Prognostic impact of an activation of coagulation in lung cancer.

BACKGROUND: There is evidence that activation of coagulation by influencing tumour biology may have impact on clinical course of lung cancer. PATIENTS AND METHODS: We measured the activation markers thrombin-antithrombin complex (TAT) and prothrombin fragment F1 + 2 in 99 lung cancer patients immediately after diagnosis, before antineoplastic treatment. Outcome was assessed at the end of appropriate standard primary therapy (four to six courses of chemotherapy, surgery or radiation). RESULTS AND CONCLUSIONS: The activation markers (means +/- SEM) were lower in the 33 responders (RSP; complete or partial remission) than in the 66 non-responders (NRSP): TAT 3.96 +/- 0.48 vs. 9.69 +/- 1.57 micrograms/l (P < 0.001), and F1 + 2 1.09 +/- 0.09 vs. 1.64 +/- 0.25 nmol/l (P < 0.05). TAT levels were > 6 micrograms/l in 30 of 66 (45%) NRSP, but only 4 of 33 (12%) RSP. 88% of patients with TAT < or = 6 micrograms/l achieved remission, and 45% with TAT > 6 micrograms/l (P = 0.0014). In the subgroup of 46 patients with advanced disease, the six RSP showed lower TAT than the 40 NRSP: 4.65 +/- 0.94 vs. 11.92 +/- 2.49 micrograms/l (P < 0.01); one of six (17%) RSP, but 21 of 40 (53%) NRSP showed TAT > 6 micrograms/l. These data suggest that in lung cancer the activation of coagulation is an independent prognostic factor, since TAT levels were different between RSP and NRSP, also within the homogeneously unfavourable metastatic subgroup. It should be further studied, whether TAT can identify patients, whose prognosis could be improved by anticoagulation as an adjunct to standard antineoplastic therapy.

Supportive pentoxifylline in falciparum malaria: no effect on tumor necrosis factor alpha levels or clinical outcome: a prospective, randomized, placebo-controlled study.

Pentoxifylline (POF) may suppress overproduction of tumor necrosis factor alpha (TNF alpha), which is thought to contribute to complications of human falciparum malaria. However, POF is believed to improve impaired capillary blood flow, which can be impaired in falciparum malaria. To test whether POF affects TNF alpha serum levels or other variables in this disease, we administered POF (20 mg/kg/day intravenously in 150 ml of saline for five days) randomized versus placebo (150 ml of saline without POF) in addition to standard antimalarial therapy. After recruitment of 51 patients with Plasmodium falciparum malaria, those receiving POF had more nausea and abdominal discomfort than the placebo group, as expected. Eleven of 27 patients receiving POF and three of 24 patients receiving placebo requested termination of the study medication (P < 0.05). Pentoxifylline did not change the decrease of TNF alpha levels or affect the clinical course in a significant way. Since POF failed to improve the clinical situation or to impact numerous laboratory parameters (including TNF alpha, thrombin-antithrombin III, thrombomodulin, and human neutrophil elastase), the study was terminated earlier than planned. While this study does not specifically address cerebral complications of malaria, the results suggest that POF is not useful as a routine adjunct to the standard therapy of falciparum malaria.

Four novel mutations in deficiency of coagulation factor XIII: consequences to expression and structure of the A-subunit.

The characterization of naturally occurring mutations is one way to approach functionally significant domains of polypeptides. About 10 mutations have been reported in factor XIII (FXIII) A-subunit deficiency, but very little is known about the effects of the mutations on the expression or the structure of this enzyme. In this study, the recent crystallization of FXIII A-subunit and determination of the three-dimensional model were used for the first time to pursue the structural consequences of mutations in the A-subunit. The molecular analysis of four families from Sweden, Germany, and Denmark revealed four previously unreported point mutations. Three of the mutations were missense mutations, Arg326-->Gln, Arg252-->Ile, and Leu498-->Pro, and one was a nonsense mutation, a deletion of thymidine in codon for Phe8 resulting in early frameshift and premature termination of the polypeptide chain. In the case of the nonsense mutation, delT Phe8, the steady-state mRNA level of FXIII A-subunit was reduced, as quantitated by reverse transcriptase-polymerase chain reaction and solid-phase minisequencing. In contrast, none of the missense mutations affected mRNA levels, indicating the possible translation of the mutant polypeptides. However, by enzyme-linked immunosorbent analysis and immunofluorescence, all the patients demonstrated a complete lack of detectable factor XIIIA antigen in their platelets. In the structural analysis, we included the mutations described in this work and the Met242-->Thr mutation reported earlier by us. Interestingly, in the three-dimensional model, all four missense mutations are localized in the evolutionarily conserved catalytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do not affect any of the residues known to be directly involved in the enzymatic reaction. The structural analyses suggest that the mutations are most likely interfering with proper folding and stability of the protein, which is in agreement with the observed absence of detectable FXIIIA antigen. Arg326, Arg252, and Met242 are all buried within the molecule. The Arg326-->Gln and Arg252-->Ile mutations are substitutions of smaller, neutral amino acids for large, charged residues. They disrupt the electrostatic balance and hydrogen-bonding interactions in structurally significant areas. The Met242-->Thr mutation is located in the same region of the core domain as the Arg252-->Ile site and is expected to have a destabilizing effect due to an introduction of a smaller, polar residue in a tightly packed hydrophobic pocket. The substitution of proline for Leu498 is predicted to cause unfavorable interatomic contacts and a disruption of the alpha-helix mainchain hydrogen-bonding pattern; it is likely to form a kink in the helix next to the dimer interface and is expected to impair proper dimerization of the A-subunits. In the case of all four missense mutations studied, the knowledge achieved from the three-dimensional model of crystallized FXIII A-subunit provides essential information about the structural significance of the specific residues and aids in understanding the biologic consequences of the mutations observed at the cellular level.

Factor XIII deficiency: pathogenic mechanisms and clinical significance.

Congenital factor XIII deficiency is a rare disease, but has provided valuable information on the physiological role of factor XIII and the benefit of factor XIII replacement therapy. It could be shown that not only homozygous patients but also heterozygotes are at risk for bleeding complications. Acquired factor XIII deficiency, however, is much more common, and preliminary studies suggest a lack of factor XIII to be an important feature of various diseases. In acute states and severe hemorrhages, replacement therapy with factor XIII concentrates is recommended. Recent progress in assay methods and future clinical studies should help to evaluate the therapeutic potential of factor XIII.

The role of inflammatory cells and their proteases in extravascular fibrinolysis.

Extravascular fibrin formation and dissolution is a pivotal event in numerous inflammatory and malignant diseases. In inflammatory cells such as monocytes/macrophages, neutrophil granulocytes appear to interact intimately with hemostasis and regulate the activity of the cascade systems of coagulation and fibrinolysis. Proteases such as neutrophil elastase are thought to influence components of hemostasis, and furthermore provide an alternative pathway of fibrinolysis. Histological, experimental, and clinical data suggest that extravascular fibrinolysis, mediated both by the plasmin system and by proteases like neutrophil elastase, is a prominent finding in various diseases such as lung cancer, chronic inflammatory bowel disease, vasculitis and connective tissue disease, bacterial sepsis, and septic shock.

Alpha-Chain cross-linking in fibrin(ogen) Marburg.

The fibrinogen structural variant, Marburg (A alpha 1-460B beta gamma)2, is comprised of normal B beta and gamma chains but contains severely truncated A alpha chains that are missing approximately one half of their factor XIIIa cross-linking domain. Immunochemical studies of fibrin(ogen) Marburg were conducted to characterize the degree to which deletion of a defined A alpha-chain segment, A alpha 461-610, can affect the process of fibrin stabilization, ie, the factor XIIIa-mediated covalent interaction that occurs between alpha chains of neighboring fibrin molecules and between alpha chains and alpha 2 antiplasmin (alpha 2PI). The ability of Marburg (and control) alpha chains to serve as a substrate for factor XIIIa and undergo cross-linking was examined in an in vitro plasma clotting system. The capacity for alpha-chain cross-linking was evaluated both as the covalent incorporation of the small synthetic peptide, NQEQVSPLTLLK (which represents the first 12 amino acids of alpha 2PI and includes the factor XIIIa-sensitive glutamine residue responsible for the cross-linking of alpha 2PI to fibrin), and as the appearance of native (ie, natural), high-molecular-weight, cross-linked alpha-chain species. Antibodies specific for the (A)alpha and gamma/gamma-gamma chains of fibrin(ogen) and for the peptide and its parent protein, alpha 2PI (68 kD), were used as immunoblotting probes to visualize the various cross-linked products formed during in vitro clotting. Recalcification of Marburg plasma in the presence of increasing concentrations of peptide resulted in the formation of peptide-decorated Marburg alpha-chain monomers. Their size at the highest peptide concentration examined indicated the incorporation of a maximum of 3 to 4 mol of peptide per mole of alpha-chain. In the absence of alpha 2PI 1-12 peptide, the alpha chains of Marburg fibrin cross-linked to form oligomers and polymers, as well as heterodimers that included alpha 2PI. Both the peptide-decorated monomers and the native cross-linked alpha-chain species of Marburg fibrin were smaller than their control plasma counterparts, consistent with the truncated structure of the parent Marburg A alpha chain. Collectively, the findings indicate that, although deletion of the A alpha chain region no. 461-610 in fibrinogen Marburg prevents formation of an extensive alpha polymer network (presumably due to the absence of critical COOH-terminal lysine residues), it does not interfere with initial events in the fibrin stabilization process, namely, factor XIII binding and the ability of alpha chains to undergo limited cross-linking to one another and to alpha 2PI.

Pharmacokinetics and tolerability of factor XIII concentrates prepared from human placenta or plasma: a crossover randomised study.

The pharmacokinetics and tolerability of factor XIII (FXIII) from plasma were compared with those of FXIII from placenta in a randomised, double-blind, crossover study involving 13 patients with congenital FXIII deficiency. Both FXIII activity and FXIII antigen were monitored. No difference was seen in the mean half-lives of the two preparations (9.3 days and 9.1 days for plasma and placenta FXIII activity, respectively). Response was similar for both preparations, but was slightly greater for FXIII from plasma (1.6 ormula: see text] vs 1.5 [formula: see text]). Similar results were found for recovery (65% vs 60%). The area under the data completed by extrapolation was significantly higher for FXIII from plasma. No differences between preparations in terms of efficacy or tolerability were observed. It can be concluded that treatment with FXIII concentrate from plasma is as efficient as with FXIII concentrate from placenta in terms of recovery and half-life. Both preparations were equivalent in terms of safety during the observation period. With the administration of monthly injections of approximately 30 U/kg serious bleeding events were prevented and no other serious adverse events occurred.

D-dimer tests detect both plasmin and neutrophil elastase derived split products.

Both plasmin and elastase, a protease released from neutrophil granulocytes, are known to degrade fibrin(ogen). This raises the possibility that elevated plasma levels of split products such as D-dimer may in part result from elastase action. After incubation in vitro of fibrinogen and fibrin clots with elastase, a clearcut increase of D-dimer immunoreactivity was demonstrated by two commercial ELISA kits. In the plasma of 79 patients with inflammatory bowel disease, D-dimer values measured by one of the ELISA kits were correlated significantly not only with markers of thrombin and plasmin activation, but also with elastase-alpha 1-antitrypsin complexes (r = 0.3555; P = 0.014). Thus, the findings of this study suggest that indeed the D-dimer levels in patients with inflammatory disorders are influenced by neutrophil elastase. New tests discriminating effects of activated haemostasis from proteolysis by neutrophil enzymes might be helpful in differential diagnosis and monitoring of therapy.

Coagulation factors and markers of activation of coagulation in homocystinuria (HOCY): a study in two siblings.

Homocystinuria due to cystathionine-beta-synthase deficiency (CBS-def-HOCY) initially often present with thromboembolic events. In most cases in which coagulation factors have been analysed, a deficiency of AT-IIIc and factor VIIc has been reported, the cause of which has not been elucidated. Activation of coagulation with consumption of coagulation factors has been postulated as the mechanism. This paper reports a longitudinal study of two patients: patient 1 with thromboembolic disease and his asymptomatic sister, patient 2. Before start of therapy in patient 1, a reduction of FVIIc, other coagulation factors, and AT-IIIc was found. Markers of activation of coagulation (F1 + 2, TAT, FM, D-dimers) were elevated only in patient 1, and only at the time of thrombotic complications. In patient 2 reduced levels of FVIIc and other coagulation proteins, and a low borderline AT-IIIc level was found. Thus, in the two patients, sustained activation of coagulation can be reasonably excluded to be the cause of low levels of coagulation proteins. Vitamin therapy with 15 mg folate and 600 mg pyridoxine per day led to almost complete normalization of amino acids in urine and plasma. Thrombosis has not recurred to date. FVIIc and the other coagulation proteins and AT-IIIc increased in parallel with the biochemical remission. Direct inhibition of the activity of AT-III and coagulation factor VIII and other factors by homocysteine was attempted in vitro but could not be shown at HC concentrations known to occur in the plasma of HOCY patients. Therefore, in these patients, deficient synthesis of coagulation factors and AT-III due to a disturbance of amino acid metabolism is still the most probable explanation for the observed low levels.

Ulcerative colitis and Crohn's disease: factor XIII, inflammation and haemostasis.

An important role has been ascribed to plasma factor XIII (FXIII) in inflammation and wound healing. FXIII is necessary for fibrin stabilization and interacts with connective tissue and adhesive proteins and cells. In a prospective study, FXIII activity and parameters of coagulation, fibrinolysis and inflammation, were determined in patients with ulcerative colitis (UC; 13 active, 22 moderate) and Crohn's disease (CD; 36 active, 45 moderate). FXIII levels were lower in active than in moderate UC and CD, and were < 70% of normal values in 7/13 patients with active UC, and in 7/36 patients with active CD, although the median values did not fall below the normal range. FXIII was somewhat higher in active UC patients responding to therapy. The FXIII levels were widely scattered, and low values appear to be due to greatly enhanced turnover. A correlation between FXIII and the systemic levels of markers of activation of haemostasis and inflammation was lacking, but there was a correlation with the extent of bowel involvement. FXIII levels were lower in the patients with involvement beyond the sigmoid colon in CU (p = 0.0045), and both small and large bowel segments in CD (p = 0.0223) patients. This points to local consumption and/or loss of FXIII within the inflamed tissue, and provides an argument for FXIII substitution in the treatment of acute episodes of inflammatory bowel diseases.

Protein S degradation in vitro by neutrophil elastase.

Human protein S is degraded by neutrophil elastase. The characteristics of cleavage are compared in a purified protein S preparation, a concentrate of vitamin K-dependent proteins (PPSB) and in normal plasma as well as in alpha-proteinase inhibitor (alpha PI)- deficient plasma. Elastase incubation of purified human protein S (molar enzyme-to-substrate-ratio 1:5500-1:55) reduces the molar mass of the native protein S (81-83 kDa) to about 79 kDa by cleavage of a small peptide. Incubation with very high elastase concentrations (molar enzyme-to-substrate-ratio 1:5.5) completely degrades protein S into small fragments. The elastase incubated protein S has a higher isoelectric point than the native form (Ip 5.9 vs. 5.3). Protein S in a PPSB coagulation factor concentrate is degraded in the same way as isolated protein S. By immunoblotting also smaller split products of molar masses between 34 and 70 kDa are demonstrated. In normal plasma protein S is not degraded by elastase concentrations up to 14 mumol l-1. In plasma of a patient with alpha 1-proteinase inhibitor deficiency protein S can be degraded by elastase. The native 82 kDa protein is degraded to a 72 kDa protein. PEG precipitation of the protein S- C4b- binding protein-complex shows that elastase predominantly splits the free protein S.

Activation of coagulation and fibrinolysis in patients with lung cancer: relation to tumour stage and prognosis.

Activation of coagulation and fibrinolysis within tumour tissues is thought to be associated with tumour growth, angiogenesis, and metastasis. The plasma levels of markers of thrombin and plasmin generation are sensitive tools for monitoring activation of coagulation and fibrinolysis. We studied 47 patients with histologically confirmed lung cancer, 15 with small cell (SCLC) and 32 with non-small cell lung cancer (NSCLC). The plasma levels of the following markers were assessed:thrombin-antithrombin III complex (TAT), prothrombin activation fragment F1 + 2, plasmin-alpha 2-antiplasmin complex (PAP) and the split product from cross-linked fibrin, D-dimer. The first sample was obtained before receiving any specific antineoplastic treatment. The patients were followed thereafter until treatment was terminated. There was no difference in activation markers between patients with SCLC and NSCLC. Comparing patients with limited disease to those with extensive disease, there were significant differences in TAT (median 3.0 (1.9-9.8) vs 5.3 (1.8-35.6) micrograms/l,P = 0.021) and D-dimer (569 (135-1948) vs 1288 (120-2221) micrograms/l, P = 0.014). According to the response to subsequent treatment, those who achieved complete or partial tumour remission had significantly lower baseline levels samples than non-responders (TAT 2.9 (1.9-4.0) vs 4.7 (1.8-35.6) micrograms/l,P = 0.0047;D-dimer 527 (135-1149) vs 1242 (120-2221) micrograms/l, P = 0.0013). Thus, the increase of TAT and D-dimer appears to be related to tumour spread. The results suggest that high levels of these markers might be a sign of unfavourable prognosis in patients with lung cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Activators of coagulation in cultured human lung-tumor cells.

Tumor matrix generation and tumor cell growth are supported by coagulation processes within the tumor tissue. Activators of coagulation were searched for in suspensions of 9 permanent human squamous-cell lung-cancer (EPLC 32MI, U1752), large-cell lung-cancer (LCLC 97TMI, LCLC 103H, U1810), and small-cell lung-cancer (N-592, H-526, DMS79, 86MI) cell lines. Incubation with these cells shortened the recalcification time in normal plasma (also in the presence of antibodies against tissue factor) or coagulation-factor-VII-, VIII-, IX- or X-deficient plasmas. The activators of coagulation in the 2 most active cell lines (U1752 and LCLC 103H) were further characterized in purified systems: the cleavage of chromogenic substrates, and the generation of markers of pro-thrombin activation were assessed. Three activators of coagulation were found in intact or sonicated cell suspensions and culture supernatants: (i) a tissue factor (TF)-like activity; (ii) an activity activating factor X, which in contrast to "cancer pro-coagulant" was not inhibited by iodoacetamide; and (iii) an activity-activating pro-thrombin, which was inhibited by the serine protease inhibitor PMSF and appeared to require plasmatic co-factor(s). The heterogeneous expression of coagulation activators by lung-tumor cell lines might be of significance for tumor biology and response to therapy.

Presence of anticardiolipin antibodies discriminates between Wegener's granulomatosis and microscopic polyarteritis.

ANCA-tests are not always useful in evaluation of disease activity of patients with Wegener's granulomatosis (WG) and microscopic polyarteritis(MP). If ANCA-tests are failing in patients with Wegener's granulomatosis or microscopic polyarteritis, markers of activated coagulation are helpful in evaluation of disease activity. The measurement of anticardiolipin antibodies(ACA) may allow to discriminate between MP and WG, since ACA are only significantly elevated in patients with MP. Anticardiolipin antibodies (ACA) are acquired autoantibodies that are found in patients with a wide spectrum of clinical conditions, mainly systemic lupus erythematosus and related disorders. They are associated with increased thrombotic tendency. We investigated in patients with ANCA-positive vasculitis whether membrane phospholipids exposed in injured endothelial cells might stimulate the formation of ACA, thus contributing to the vascular thrombotic occlusion besides the activation of coagulation.

Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids A alpha 461-610 (Lys 461 AAA-->stop TAA).

In the A alpha-chain gene coding for an abnormal fibrinogen (fibrinogen Marburg) we identified a single base substitution (A-->T) that changes the codon A alpha 461 AAA (Lys) to TAA (Stop). The propositus was found to be homozygous for the mutation, whereas the father and five siblings were heterozygous, and three other siblings contained only the normal sequence. The stop codon at position 461 results in the deletion of the carboxyl-terminal segment A alpha 461-610. Purified fibrinogen Marburg contained an A alpha-chain with a relative molecular weight of approximately 47,000. The FpA release by thrombin was not affected by this deletion, whereas the fibrin polymerization was strongly decreased. The binding of endothelial cells to immobilized fibrinogen Marburg was almost completely abolished compared with normal fibrinogen. Fibrinogen Marburg contained a substantial amount of albumin linked to the fibrinogen molecule by disulfide bonds, and these fibrinogen-albumin complexes were also present in plasma. The plasma fibrinogen concentration of the propositus was measured by three different methods: a functional method (< 0.25 mg/mL), an immunologic method using polyclonal antibodies (0.6 mg/mL), and an immunologic method based on two monoclonal antibodies specific for the amino-terminus and carboxyl-terminus of the A alpha-chain (< 0.05 mg/mL). Using the two immunologic methods, it appeared that only 10% to 15% of the plasma fibrinogen of the heterozygous siblings was abnormal.

Protein C degradation in vitro by neutrophil elastase.

Purified protein C is completely degraded into small peptides by in vitro incubation with purified elastase. Protein C is a rather sensitive substrate as degradation is already accomplished by low elastase concentrations (molar enzyme-to-substrate ratio 1:510) and short incubation periods (5 min-60 min). Protein C in a PPSB coagulation factor concentrate is equally degraded and similar split products are detected by blotting techniques. The protein C activity (measured by a chromogenic substrate) is faster reduced by elastase than the protein C concentration (measured by an ELISA). Incubation of normal plasma with high elastase concentrations (5.7 nmol/ml plasma) results in reduction of the protein C band while no split products are detectable. The pathophysiologic significance of the effects of elastase on protein C remains to be elucidated.