Oversulfated heparin by-products induce thrombin generation in human plasmas through contact system activation.

Thrombin generation is thought to be mediated predominantly by the tissue factor or ''extrinsic'' coagulation pathway. An alternate pathway to thrombin generation (the ''intrinsic'' pathway or contact system) has been observed when blood or plasma comes in contact with artificial surfaces. Here we present evidence for a new route to thrombin formation that begins with the activation of the contact system protein prekallikrein by oversulfated heparin (OS-HB). Kallikrein, instead of activated factor X, cleaves prothrombin to form thrombin. Thrombin then cleaves fibrinogen to form fibrin clots. Moreover, we show that OS-HB by-products induce kallikrein- and thrombin-like activities in normal human plasma and in human plasma devoid of coagulation factor X or downstream contact system components factor IX or factor XI. Oversulfated heparin by-product-induced thrombin generation may have had a role in the adverse reactions associated with the recent clinical use of contaminated heparin.

Heparin and oversulfated heparin byproduct induce thrombin generation through contact system activation in plasma of patients with HIT.

Heparin-induced thrombocytopenia with thrombosis (HITT) is the most severe side effect of heparin administration. Patients with HITT may die or have permanent sequelae such as stroke or limb amputation. Contaminated heparin is associated with anaphylactic reactions and deaths by activating the contact system. It is also associated with high incidence of HIT via a yet unknown mechanism. This study showed that although oversulfated heparin byproduct induced thrombin activities in both normal and HIT patient plasmas through the contact system activation, authentic heparin induced thrombin activities only in HIT patient plasmas containing autoantibodies against protein/ heparin complex. These data suggest that the negatively charged immunoglobulin G (IgG)/platelet factor 4 (PF4)/heparin complex activate the contact system and produce thrombin in human plasma, and thrombin partially activates the platelets allowing subsequent platelet activation through IgG/Fc receptor II signaling. The newly discovered mechanism of heparin-induced thrombin activity could explain the increased incidence of HIT in patients exposed to contaminated heparin. Furthermore, the assays used in these studies would be valuable for HIT diagnosis, prevention, and treatment.

Characterization of the anticoagulant actions of a semisynthetic curdlan sulfate.

Sulfation of curdlan, a natural, linear beta-1,3-glucan results in potent anticoagulant and antithrombotic agents. The different activity characteristics in the classical coagulation assays were shown to depend on various structural parameters. To obtain more detailed information about their structure-dependent mechanisms of action, one representative of these beta-1,3-glucan sulfates (CurS), was further investigated using several coagulation assays and amidolytic tests with chromogenic substrates. The mode of action of CurS differs from that of heparin. CurS reduces the thrombin formation by principally inhibiting the intrinsic FXa generation. As shown by amidolytic assays, it eliminates already generated thrombin mainly by accelerating the HCII-mediated thrombin inactivation, whereas its AT-mediated anti-thrombin activity is considerably lower than that of heparin. Further, it prevents the thrombin-mediated fibrin polymerization by directly interfering with the thrombin action on fibrinogen as well as by binding to fibrinogen. Finally, CurS is capable to activate the contact system with the consequence of a potential fibrinolytic effect. In conclusion, beta-1,3-glucan sulfates do not inhibit the blood coagulation nonspecifically due to their anionic character, but in dependence on their individual structure, they interfere specifically with the coagulation process at several sites.

C1 esterase inhibitor concentrate for capillary leakage syndrome following bone marrow transplantation.

The prognosis of patients with severe capillary leakage syndrome (CLS) after bone marrow transplantation (BMT) is dismal despite aggressive use of intensive care therapy. Because the activated classical pathway of complement and relatively low levels of C1 esterase inhibitor (C1 INH) activity are known features in these patients, we evaluated the efficacy of a therapy using purified, human C1 INH concentrate. Severe CLS was defined as increase in body weight by more than 3% within 24 h combined with generalized edema, impaired hemodynamic system (tachycardia and/or decreased blood pressure), and non-responsiveness to furosemide. Of 142 patients, 22 developed severe CLS. The first seven patients whom we diagnosed with this complication were assessed as control patients. Fifteen patients with severe CLS were treated with C1 INH concentrate using a cumulative dose of 180 units/kg body wt. (initial dose: 60 units/kg, followed by two doses at 30 units/kg and four doses at 15 units/kg, every 12 h). The survival rate of patients with CLS was 57% at 1 year after BMT in the C1 INH treatment group, compared with 14% in the control group (p = 0.008). Eight of 15 treated patients are alive at a median of 9 months (range: 4-55) after BMT. The plasma levels of the complement activation parameters C4d and C5a were 3 +/- 1.1 mg/dl (mean +/- S.D.) and 0.3 +/- 0.1 microgram/l, respectively, prior to BMT, increasing to 8.2 +/- 2.1 mg/dl and 1.3 +/- 0.4 micrograms/l, respectively, at diagnosis of CLS. After infusion of C1 INH concentrate the plasma levels of C5a and C4d normalized. The activity of C1 INH rose to 139 +/- 10% of normal human plasma NHP pool (mean +/- S.D.) after infusion. The CH50 values were not significantly altered. The fluid status normalized within 11 days in 14 of 15 treated patients. The results of this study suggest that therapy with C1 INH concentrate improves the prognosis of patients with CLS after BMT. This has to be confirmed in a randomized, controlled trial.

Activation of plasma kinin system correlates with severe coagulation disorders in patients with ovarian hyperstimulation syndrome.

The aim of this study was to evaluate status of plasma kinin system in patients with ovarian hyperstimulation syndrome (OHSS), in order to investigate whether activation of the plasma kinin system correlates with increased blood coagulability. In the first part of the study, concentrations of plasma prekallikrein (PK) in OHSS cycles (n = 13) were monitored from the day of human chorionic gonadotrophin (HCG) administration to the mid-luteal phase, and were compared with those of control cycles (n = 17). The average value of PK in OHSS cycles began to decrease on day 8, and by day 10 was significantly lower than that of control cycles (86 +/- 6 versus 106 +/- 4%, P <0.01). The time course of changes in PK concentration correlated well with the clinical condition of OHSS patients. In the second part of the study, we obtained data from 26 patients who were hospitalized because of severe OHSS, to investigate the correlation between PK and other haemostatic markers. OHSS patients with severe PK reduction (<80% normal, n = 9) demonstrated significantly higher values of plasma thrombin-antithrombin III and plasmin-alpha2 antiplasmin, and more severe haemoconcentration, compared to those OHSS patients who had no reduction in PK (n = 17). In conclusion, our data suggest that activation of the plasma kinin system occurs specifically and occasionally in OHSS patients, and is associated with increased blood coagulability. Thus, when an OHSS patient demonstrates a low value of plasma PK, more careful management is required to prevent thromboembolic complications.

Homozygous type I plasminogen deficiency.

Homozygous type I plasminogen (Plg) deficiency has not been described in human subjects so far. Ligneous conjunctivitis is a rare and unusual form of chronic pseudomembranous conjunctivitis of unknown etiology. Here we report for the first time on homozygous type I Plg deficiency in three unrelated female patients who suffered from ligneous conjunctivitis and additional pseudomembranous lesions of other mucous membranes. The disease is caused by massive fibrin depositions within the "extravascular space" of mucous membranes because of absent clearance by plasmin. Infusions of albumin, fresh frozen plasma, or Lys-plasminogen (Lys-Plg) into two of the three patients revealed normal Plg activation capacity in these patients. The absence of fibrinolytic activity could therefore be shown to be due to Plg deficiency. Similar studies in the third patient have not been completed. In the two patients studied so far, infusions of Lys-Plg resulted in prompt and adequate Plg recovery with a short half-life and high amounts of plasmin-antiplasmin complexes and D-dimer. One patient additionally revealed an inherited partial factor XII deficiency. Functionally, this factor XII deficiency did not interfere with Plg activation. However, there may be a pathway of Plg activation in this patient via the prekallikrein C1-INH system.

Structure/function analysis of human factor XII using recombinant deletion mutants. Evidence for an additional region involved in the binding to negatively charged surfaces.

The binding site of human factor XII (FXII) for negatively charged surfaces has been proposed to be localized in the N-terminal region of factor XII. We have generated two recombinant factor XII proteins that lack this region: one protein consisting of the second growth-factor-like domain, the kringle domain, the proline-rich region and the catalytic domain of FXII (rFXII-U-like), and another consisting of only 16 amino acids of the proline-rich region of the heavy-chain region and the catalytic domain (rFXII-1pc). Each recombinant truncated protein, as well as recombinant full-length FXII (rFXII), were produced in HepG2 cells and purified by immunoaffinity chromatography. The capability of these recombinant proteins to bind to negatively charged surfaces and to initiate contact activation was studied. Radiolabeled rFXII-U-like and, to a lesser extent, rFXII-lpc bound to glass in a concentration-dependent manner, yet with lower efficiency than rFXII. The binding of the recombinant proteins was inhibited by a 100-fold molar excess of non-labeled native factor XII. On native polyacrylamide gel electrophoresis, both truncated proteins appeared to bind also to dextran sulfate, a soluble negatively charged compound. Glass-bound rFXII-U-like was able to activate prekallikrein in FXII-deficient plasma (assessed by measuring the generation of kallikrein-C1-inhibitor complexes), but less efficiently than rFXII, rFXII-U-like and rFXII-lpc exhibited coagulant activity, but this activity was significantly lower than that of rFXII. These data confirm that the N-terminal part of the heavy-chain region of factor XII contains a binding site for negatively charged activating surfaces, and indicate that other sequences, possibly located on the second epidermal-growth-factor-like domain and/or the kringle domain, contribute to the binding of factor XII to these surfaces.

Plasmin-mediated activation of contact system in response to pharmacological thrombolysis.

BACKGROUND: Thrombin activity increases in patients treated with coronary thrombolysis for acute myocardial infarction, but the mechanisms are not well defined. We have shown that thrombin activity increases in plasma and whole blood incubated with plasminogen activators and appears to be plasmin mediated and dependent on activity of the factor VIIIa/IXa complex. METHODS AND RESULTS: In the present study, increases in thrombin activity induced by incubation of recalcified citrated plasma with 0.16 to 0.5 mumol/L plasmin at 37 degrees C were markedly attenuated in recalcified citrated plasma deficient in factors XI or XII, prekallikrein, or high molecular weight kininogen, as well as in plasma incubated with plasmin in the presence of 3.5 mumol/L corn trypsin inhibitor, a specific factor XIIa inhibitor. Increases in thrombin activity also occurred in nonanticoagulated whole blood incubated with pharmacological concentrations of plasminogen activators and were markedly attenuated in the presence of corn trypsin inhibitor. Plasmin-mediated (0.25 mumol/L) activation of purified factor XII occurred in 0.05 mol/L Tris-HCl and 0.012 mol/L NaCl (pH 7.8) at 37 degrees C, resulting in equimolar quantities of two fragments that corresponded to cleavage of factor XII at Arg353-Val354, the site involved in kallikrein-mediated activation of factor XII, and cleavage at Lys346-Ser347, an apparently novel site of plasmin-mediated hydrolysis of factor XII. Contact activation was also demonstrated in plasma samples from patients after treatment with fibrinolytic agents for myocardial infarction, by demonstrating cleavage of high molecular weight kininogen from its one-chain to its two-chain form by ligand blotting with 125I-prekallikrein. CONCLUSIONS: Plasmin-mediated activation of the contact system of coagulation appears to account, at least in part, for increases in procoagulant activity in patients treated with fibrinolytic agents. It may also explain hypotension, by release of bradykinin from high molecular weight kininogen, and complement activation, by activated factor XII, that has been demonstrated in these patients.

[An experimental model of depletion of the blood kallikrein-kinin system]. / Eksperimental'naia model' istoshcheniia kallikrein-kininovoi sistemy krovi.

A new way of kallikrein-kinin system exhaustion in experiments on animals is suggested. This approach consists in a simultaneous rise in the activity of kinin formation and destruction. It is shown that insignificant activation of the kallikrein system and kininase activity leads to activation of the kinin system and rise in active kinin levels. A 1.5-3-fold increase in the total activity of kallikrein and a simultaneous 3-6-fold increase in kininase activity cause exhaustion of the blood kallikrein-kinin system in 2 hours and makes active kinin formation impossible by the body's requirements when the synthesis of kinin system components is maintained.

Stimulation of plasmin activity in cultured human fibroblast cells by Porphyromonas endodontalis.

1. Plasmin activity in the conditioned medium of Gin-1 cells, a human gingival fibroblast cell line, was stimulated by Porphyromonas endodontalis, a putative pathogen of oral submucous abscesses, in a time- and dose-dependent manner. 2. P. endodontalis stimulated the activity of plasminogen activator in both the conditioned medium and the cell lysate. The plasminogen activator in Gin-1 cells was approx. 50 kDa by zymography. 3. The conditioned medium of Gin-1 cells exposed to P. endodontalis stimulated the conversion of human serum prekallikrein to kallikrein. 4. These results suggested that P. endodontalis stimulates the plasminogen activator-plasmin system in Gin-1 cells, and that activated plasmin plays a role in the progress of periodontal tissue inflammation.

Contact factor mediated fibrinolysis is increased by the combined oral contraceptive pill.

OBJECTIVE: To study the fibrinolytic pathways and their relationship with the contact system in women using combined oral contraceptives (COCs). DESIGN: Serial plasma samples were collected from 18 women before treatment with COCs containing 30 micrograms oestrogen during treatment cycles 3 and 6, and 2 weeks after stopping treatment. Fibrinolysis was measured before and after dextran sulphate mediated contact activation using fibrin plates. RESULTS: Fibrinolysis increased significantly during cycles 3 and 6 (from 77% to 100% and 113%, respectively, P < 0.01) and showed a further increase after dextran sulphate activation (from 134% to 158% and 167%, respectively, P < 0.01). Tissue-plasminogen activator, urokinase-plasminogen activator and plasminogen activator inhibitor did not change significantly. There were significant elevations of Factor XII (from 0.92 u/ml to 1.43 u/ml, P < 0.01) and prekallikrein (0.94 u/ml to 1.10 u/ml, P < 0.05) in cycle 3, which both remained high at cycle 6 (P < 0.01) and decreased after stopping the COC. Alpha-2-macroglobulin and C1-esterase inhibitor showed no significant change, but alpha-1-antitrypsin increased from 0.85 u/ml to 1.11 u/ml by cycle 3 (P < 0.01), and returned to near normal levels after stopping the COC. CONCLUSIONS: The increase in fibrinolysis may be due to increased levels of Factor XII and prekallikrein without a corresponding increase in their natural inhibitors (C1-esterase inhibitor and alpha-2-macroglobulin). A parallel increase in the intrinsic pathway of coagulation may be limited by elevated alpha-1-antitrypsin at the level of activated Factor XI. The increase in fibrinolysis caused by oral contraceptives may balance any potential thrombotic risk due to increased fibrinogen or vitamin K dependent coagulation factors.

Level of plasma prekallikrein and its inhibitors in reactors and nonreactors during intravenous enhancement with contrast media.

Complex contact activation systems may play a major role in the side effects of i.v. contrast media (CM). This is why quantitative measurements of several factors (plasma prekallikrein, hematocrit (hct), alpha-2-macroglobulin, alpha-1-antitrypsin, and C1-esterase inhibitor) were determined prior to and following the injection of CM during body CT examination in 5 patient groups, each (n = 10) receiving one of 5 different CM, including ioxaglate, meglumine iodamide, metrizamide, iohexol, and meglumine diatrizoate. The initial plasma prekallikrein level was available from 45 patients and was statistically lower in reactors (mean 90.6 mumol TAMe/ml/h; n = 13) than in nonreactors (mean 107 mumol TAMe/ml/h; n = 32) (p = 0.006), but there was no statistically significant difference in the decrease of plasma prekallikrein before and at 5 min after the injection for those 2 groups. The initial plasma C1-esterase inhibitor level was lower in reactors, while the plasma alpha-2-macroglobulin level was higher in that group than in nonreactors. The results indicate that the measurement of plasma prekallikrein combined with plasma C1-esterase inhibitor and alpha-2-macroglobulin measurement could be useful when predicting which patients are prone to CM reactions.