Nils U. Bang

Role of Plasminogen-Activator Inhibitor Type 1 in the Pathogenesis and Outcome of the Hemolytic Uremic Syndrome

Abstract Background Deposition of fibrin in glomeruli and renal failure are characteristic features of the hemolytic uremic syndrome. An inhibitor of glomerular fibrinolysis has been detected in plasma from children with this disorder. In this study, we define the inhibitor and show that its plasma level is correlated with the outcome of the disease. Methods and Results Plasminogen-activator inhibitor type 1 (PAI-1) in plasma was measured with an assay employing a specific monoclonal antibody in 40 consecutive children hospitalized with the hemolytic uremic syndrome: 12 who recovered adequate renal function (serum creatinine, ≤2.0 mg per deciliter [111 μmol per liter]) without dialysis, 23 who recovered adequate renal function after peritoneal dialysis, and 5 who did not recover adequate renal function after undergoing dialysis. At presentation, plasma PAI-1 levels were higher in the patients with the hemolytic uremic syndrome than in nine children with other forms of acute renal failure. That the inhibit...

Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens.

The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombins stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.

Abnormal platelet function in Chediak-Higashi syndrome.

Platelets in an infant with Chediak‐Higashi (C‐H) syndrome without bleeding manifestations and not in the accelerated phase showed abnormal function consistent with storage pool disorder as shown by abnormal aggregation, decreased storage capacity and release of [14C]5‐HT, low endogenous 5‐HT, reduced ATP and ADP with an increased ATP/ADP ratio, increased specific radioactivity of ADP after [14C]adenine labelling, decreased release of adenine nucleotides after stimulation, impaired secretion of acid hydrolases despite normal stores, and decreased calcium content. Incorporation of [14C]adenine into metabolic pool adenine nucleotides was normal. Nucleotide conversion to hypoxanthine in stimulated platelets was mildly impaired. Platelet cyclic‐AMP (c‐AMP) was initially elevated, but even when c‐AMP returned to normal levels after ascorbate treatment, platelet function was not improved. Elevated intracellular c‐AMP was not solely responsible for the abnormal platelet function.

Plasma inhibitor of glomerular fibrinolysis in the hemolytic-uremic syndrome

To detect an inhibitor of glomerular fibrinolysis, dilutions of human plasma were incubated on microscope slides with two frozen sections of normal human kidney. The slides were studied by the fibrin slide technique. The lysis inhibitory titer was defined as the highest dilution completely inhibiting glomerular fibrinolysis. Of 27 children without renal disease, none had a lysis inhibitory titer greater than 1:2. Defining an elevated lysis inhibitory titer as 1:8 or greater, we found an elevated lysis inhibitory titer in plasma from all 17 children with hemolytic-uremic syndrome. No correlation was found between the lysis inhibitory titer and the hematocrit, white blood cell or platelet counts, serum creatinine level, or levels of the antiplasmins alpha 1-antitrypsin, alpha 2-macroglobulin, C1-esterase inhibitor, or alpha 2-antiplasmin. The inhibitor was found to have a molecular weight of less than 12,000. A close correlation was discovered between the duration of lysis inhibitory titer elevation and the clinical course; removal of the inhibitor from the plasma by peritoneal dialysis was associated with improvement in renal function. Results suggest that the inhibitor may play an important role in the pathogenesis and persistence of glomerular fibrin deposition.

Prospective Study Correlating Fibrinopeptide A, Troponin I, Myoglobin, and Myosin Light Chain Levels With Early and Late Ischemic Events in Consecutive Patients Presenting to the Emergency Department With Chest Pain

BackgroundAlthough thrombus formation plays a major role in acute coronary syndromes, few studies have evaluated a thrombus marker in risk stratification of patients with chest pain. Furthermore, the relation between markers that reflect myocardial injury and thrombus formation that may predict events in a heterogeneous patient population is unknown. This study correlated markers of thrombus and myocardial injury with early and late ischemic events in consecutive patients with chest pain. Methods and ResultsSerum troponin I (TnI), myoglobin, and myosin light chain levels were obtained from 247 patients and urinary fibrinopeptide A (FPA) from 178 of the 247. By multivariate analysis, patients with an elevated FPA level were 4.82 times more likely to die or have myocardial infarction, unstable angina, and coronary revascularization at 1 week (P =0.002, 95% CI 1.78, 13.03), whereas those with an elevated TnI (>0.2 ng/mL) were 9.41 times more likely (P <0.001, 95% CI 2.84, 31.17). At 6 months (excluding the index event), an elevated FPA level was an independent predictor of events, with an odds ratio of 9.57 (P <0.001, C1 3.29, 27.8), and was the only marker to predict a shorter event-free survival (P <0.001). The other markers did not independently correlate with cardiac events, although MLC incrementally increased early predictive accuracy in combination with the FPA and TnI. ConclusionsElevated FPA and TnI correlated with cardiac events during the initial week in patients presenting to the Emergency Department with chest pain. FPA predicted adverse events and a shorter event-free survival at 6 months.

Biological Properties of a Kringleless Tissue Plasminogen Activator (t-PA) Mutant

Abstract We have previously described a recombinant t-PA mutant (mt-PA) which lacks the double kringle domains. Purified mt-PA (specific activity comparable to melanoma cell derived t-PA (nt-PA)) exhibits fibrin specificity similar to nt-PA but is inhibited 50 to 80% less than nt-PA by a human platelet PA inhibitor. The biological properties of mt-PA, nt-PA and urokinase (u-PA) were studied in a human plasma circulation system. [125I] fibrinogen whole blood Chandler loop thrombi were suspended in a chamber perfused by plasma (50 ml) circulated at 120 ml/min. The plasminogen activators were given as a bolus (20% of the total dose) and followed by a 4 h constant rate infusion. Samples obtained were analysed for [125I] fibrin degradation products, plasminogen and fibrinogen. At 5h, thrombolysis (% total thrombus [125I]) released was: nt-PA (100u/ml) 51±710 (mean ±s.d.), mt-PA (100u/ml) 79±7%, u-PA (500u/ml) 88±12%. Plasminogen and fibrinogen values (% of control) were: nt-PA 63±90/ plasminogen, 68±19% fibrinogen; mt-PA 69±7% plasminogen, 72±23% fibrinogen; u-PA 34±8% plasminogen, 21±8% fibrinogen. In saline control studies, no thrombolysis and no plasminogen and fibrinogen changes occurred. Thus, mt-PA appears to be a more efficient thrombolysic agent than nt-PA; the two plasminogen activators cause comparable plasminogen and fibrinogen depletion. 100 u mt-PA is comparable to 500u u-PA/ml but as expected, u-PA extensively depletes both plasminogen and fibrinogen.

Serial Urinary 11-Dehydrothromboxane B2, Aspirin Dose, and Vascular Events in Blacks After Recent Cerebral Infarction

Background and Purpose— Incomplete platelet inhibition by aspirin (aspirin resistance) may be a reason for stroke recurrence in some patients. 11-Dehydrothromboxane B2 (11-DTB2) is a stable thromboxane A2 metabolite that reflects in vivo platelet activation. This pilot study was intended to evaluate the reproducibility of urinary 11-DTB2 over time and to look for evidence of aspirin resistance. Methods— All subjects were screened for the African American Antiplatelet Stroke Prevention Study (AAASPS) 7 to 90 days after noncardioembolic cerebral infarction. Of 83 subjects with at least 1 urine sample, 52 were enrolled in AAASPS (randomized to blinded treatment with aspirin 650 mg/d or ticlopidine 500 mg/d), and 31 were enrolled in an open-label antiplatelet therapy cohort. Subjects were followed up for 2 years, with 11-DTB2 measurements scheduled at baseline and 6, 12, and 24 months. Vascular events were cerebral infarction, myocardial infarction, or vascular death. Results— Despite considerable individual up or down fluctuations, the median 11-DTB2 change did not significantly differ from zero in any of the subgroups. However, in 6 subjects with a 4-fold decrease in aspirin dose from 1300 to 325 or 81 mg/d, the 11-DTB2 level increased from 611 to 1881 pg/mg creatinine (P =0.06). Vascular events occurred in 7 of 61 aspirin-treated subjects, and 11-DTB2 levels did not correlate with the events. Conclusions— Fluctuations in urinary 11-DTB2 after cerebral infarction in blacks do not correlate with changes in aspirin doses, except perhaps when the dose changes by a factor of 4 or more. A larger study is needed to look further for aspirin resistance.

Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin

Summary Two glycoforms of a soluble mutant of recombinant human thrombomodulin (rec.TM) were used to identify critical N‐ and O‐linked glycans of the endothelial cell thrombin receptor. While N‐linked glycans were not found to be involved in any function of rec.TM, an acidic chondroitin sulphate‐like glycosaminoglycan (CSGAG) was found to be critical for all the direct anticoagulant functions of rec.TM, including inhibition of thrombin‐mediated platelet aggregation. A glycoform of rec.TM lacking CSGAG had very poor anticoagulant activity. Furthermore, the glycoform of rec.TM possessing CSGAG showed strong inhibition by and had high affinity for poly‐cationic basic proteins, whereas the CSGAG‐deficient rec.TM did not. Monoclonal antibody binding as well as lectin mapping of rec.TM with agglutinins identified sialic acid containing O‐linked glycans in both glycoforms additional to the CSGAG in high molecular weight rec.TM. These findings define important molecular interactions modulating the anticoagulant function of TM, which appear to be critically regulated by CSGAG, and also showed that the overall post‐translational glycosylation pattern of the two glycoforms was very similar except for the presence of CSGAG. The possibility exists that differently expressed glycoforms of TM may be crucial for the expression of endothelial cell‐related anticoagulant potential in different vascular beds.

Recombinant human thrombomodulin attenuates human endothelial cell activation by human thrombin.

Two glycoforms of recombinant human thrombomodulin (TM; TMD1-105 and TMD1-75), an endothelial cell membrane protein, were tested for their ability to alter thrombin-induced activation of cultured human umbilical vein endothelial cells (HUVECs). After stimulation with 10 nmol/L thrombin, HUVEC generation of inositol-1,4,5-trisphosphate (IP3), a potent Ca(2+)-mobilizing second messenger, was dose-dependently blocked by TMD1-105. Both TMD1-105 (IC50 = 10 nmol/L) and TMD1-75 (IC50 = 100 nmol/L) blocked the enhanced prostacyclin synthesis by HUVEC monolayers treated with 10 nmol/L thrombin. HUVEC monolayer permeability to Evans blue dye-labeled albumin increased from 0.125 +/- 0.06 microL/min in control experiments to 0.380 +/- 0.09 microL/min after treatment with 100 nmol/L thrombin (P < .05). Incubation of HUVECs with TMD1-105 alone (600 nmol/L) had no effect (0.114 +/- 0.04 microL/min) on basal permeability. In contrast, incubation of 100 nmol/L thrombin with 600 nmol/L TMD1-105 reduced this increase in HUVEC permeability to almost control levels (0.142 +/- 0.06 microL/min). These results demonstrate that recombinant human TM, a potent in vitro anticoagulant, also functions as an antagonist of thrombin receptor-mediated HUVEC activation. In addition to its anticoagulant functions, the high-affinity endothelial cell receptor TM may play a role in modulating endothelial cell activation by thrombin.

MONOCYTE/MACROPHAGE‐MEDIATED CATABOLISM OF FIBRINOGEN AND FIBRIN

Traditionally, studies on fibrinogen and fibrin catabolism have focused on the humoral plasmin-mediated pathway. However, additional enzyme systems of considerable pathophysiological importance in the catabolism of fibrin deposits, as well as circulating soluble fibrin complexes, are located in cells of phagocytic potential, the polymorphonuclear leukocytes, and the monocytesmacrophages of the reticuloendothelial system. Soluble fibrin complexes (SFC) formed through either thrombin-fibrinogen interactions or limited proteolysis of noncrosslinked fibrin have been shown in several laboratories, including our own, to be removed from the circulation mainly by monocytes-macrophages of the reticuloendothelial system in the liver and spleen.’-* Our longstanding interest in SFC, which is demonstrable in large quantities in the circulation in clinical and experimental thrombotic states,5 led us to the systematic exploration of the degradation of these macromolecules by intact macrophages and macrophage enzymes reported below.