The streptokinase domain responsible for plasminogen binding

The limited proteolysis of soluble recombinant streptokinase by Sepharose-immobilized human plasmin(ogen) complexed with recombinant streptokinase is described. A 17 kDa streptokinase fragment comprising residues Val143 (Glu148) to Arg289 (Lys293) has been shown to be the smallest stable plasminogen binding domain. Moreover, this region seems also to be important for plasminogen activation.     

Discrepant changes in plasminogen by two different assays in patients receiving streptokinase

The fluorogenic synthetic substrate and radial immunodiffusion assays of plasma plasminogen were compared before and after administration of intravenous streptokinase in differing doses to 57 patients being treated for acute myocardial infarction. There was a moderate correlation (r = 0.73, slope = 0.221, intercept = 1.005, n = 57 pairs) in the two assays of plasma plasminogen before the administration of streptokinase. After streptokinase, however, the correlation of the two assays was poor (r = 0.28, slope = 0.03, y-intercept = 0.003, n = 57 pairs). The decrease in plasma plasminogen by the fluorogenic synthetic substrate assay after streptokinase averaged 95 +/- 5%, with little variation between doses. In contrast, the percentage decrease in plasma plasminogen after streptokinase by the radial immunodiffusion assay averaged only 30 +/- 11%. The percentage change in plasma plasminogen by the two assays is significantly different (P = 0.001). The discrepancy in the percentage change in plasma plasminogen after streptokinase as measured by the fluorogenic synthetic substrate assay and the radial immunodiffusion assay can be explained by a lack of specificity for the antibody to plasminogen in the radial immunodiffusion kit. Antigen-antibody precipitin rings were observed after incubation of antibody with a mixture presumed to contain plasmin, plasmin-alpha 2 antiplasmin complexes, and plasmin-fibrin/fibrinogen degradation products. Based on these data, the fluorogenic synthetic substrate assay for plasma plasminogen is a superior means of following plasminogen depletion in response to thrombolytic therapy after streptokinase treatment for acute myocardial infarction.     

Structural diversity of streptokinase and activation of human plasminogen

The beta domain of streptokinase is required for plasminogen activation and contains a region of sequence diversity associated with infection and disease in group A streptococci. We report that mutagenesis of this polymorphic region does not alter plasminogen activation, which suggests an alternative function for this molecular motif in streptococcal disease.     

Catheter-induced superior vena cava thrombosis-lysis with streptokinase and apsac (Anisoylated plasminogen streptokinase activator complex BRL 26921)

Ten patients were given thrombolytic therapy for obstructive thrombi in the superior vena cava (SVC) following catheterisation for total parenteral nutrition (TPN—8 cases) or chemotherapy (2 cases). Seven of the TPN patients were given intravenous APSAC (Beecham, BRL 26921); the other 3 were given streptokinase (SK). Five of the caval occlusions treated with APSAC cleared after between 1 and 5 doses; one of those given SK cleared after 3 days continuous infusions during which active thrombolytic potential was maintained.Intravenous APSAC should be considered for the relief of major SVC occlusive thrombosis secondary to central venous catheters.     

Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA.

Hepatocyte growth factor, also known as scatter factor, is a complete mitogen for hepatocytes that bears sequence and structural homology with plasminogen. Because it exists in both a mitogenically inactive single-chain form and an active two-chain form, we were interested in determining whether plasminogen activators could properly cleave single-chain hepatocyte growth factor to generate active two-chain hepatocyte growth factor. Herein we report that both urokinase-type plasminogen activator and tissue-type plasminogen activator can cleave single-chain hepatocyte growth factor, generating two-chain hepatocyte growth factor. When equal quantities of plasminogen activator-treated and activator-untreated hepatocyte growth factor are compared in serum-free in vitro bioassays, the treated hepatocyte growth factor is mitotically more active. Also, urokinase-type plasminogen activator was inactive against hepatocyte growth factor molecules with a mutated cleavage site. This suggests that urokinase-type and tissue-type plasminogen activator may be natural biological regulators of hepatocyte growth factor. Because the active form of hepatocyte growth factor is a powerful stimulator of DNA synthesis and cell motility, these findings may be relevant in understanding the role of plasminogen activators in the biology of cancer invasion and metastasis.     

Evidence for a continuing accumulation of APSAC (Anisoylated plasminogen streptokinase activator complex) by human clots: Comparison with the binding of other plasminogen activators and Plasminogen

The uptake of [¹²⁵I-plasminogen]-APSAC into crosslinked human plasma clots from autologous plasma was significantly greater than the uptake of equimolar iodinated lys77-plasminogen. The uptake of APSAC in the presence of 6-aminohexanoic acid (10 mM) was also significantly greater than the uptake of plasminogen in the presence of 6-aminohexanoic acid.The accumulation of APSAC continued for at least 2 h (in nonlysing conditions), was similar in platelet-rich, platelet-poor and whole-blood clots and was not affected by prior aging of the clots (up to 6 h). The uptakes of APSAC and streptokinase plasminogen were slightly affected by the plasma concentration of anti-SK IgG and, in general, the fibrin-binding of APSAC was similar to the binding of radiolabelled t-PA. Urokinase (high-molecular weight) did not demonstrate fibrin- binding, the uptake being explained by simple diffusion (quantified using albumin). The relative order of continuing accumulation APSAC = t-PA > lys-plasminogen > urokinase is similar to the order of initial binding (< 1 min incubation), and we conclude that the formation of an acylated, stabilised, activator complex of lys-plasminogen and streptokinase imparts additional fibrin-binding to lys-plasminogen.     

Tissue plasminogen activator therapy of rabbit nephrotoxic nephritis.

To study the efficacy of tissue plasminogen activator (PA) therapy to prevent deteriorating renal function in experimental proliferative glomerulonephritis we used a model of nephrotoxic nephritis induced in rabbits by injection of antiglomerular basement membrane antiserum. Saline or recombinant tissue plasminogen activator (rt-PA, 1.3 mg/kg body weight) was infused daily for 7 days starting from day 7 after the injection of antiglomerular basement membrane antiserum when the disease had been already triggered. Animals were killed on day 14. Rabbits given saline had abundant deposits of fibrin and crescents in about 67% of glomeruli. rt-PA significantly protected animals from glomerular fibrin deposition and crescent formation with respect to saline-treated animals. Renal function measured as creatinine clearance was dramatically impaired in rabbits given saline. Treatment with rt-PA ameliorated the renal function impairment of nephrotoxic nephritis. rt-PA did not produce a systemic fibrinolytic state as indicated by alpha 2-antiplasmin level measurement. These results suggest that tissue PA may have important implications in preventing renal function deterioration in humans with crescentic glomerulonephritis and fibrin depositions.     

Detection of nephritis strain-associated streptokinase by monoclonal antibodies

Monoclonal antibodies (MAbs) N-59 and RU-1 were produced by immunisation of mice with streptokinase secreted by Streptococcus group A, type 12, strain A374 isolated from a patient with post-streptococcal glomerulonephritis (PSGN) and were characterised by Western blot analysis. MAb N-59 recognised antigenic determinants shared by both nephritis strain-associated streptokinase (NSA-SKase) and streptokinase of Streptococcus group C (C-SKase); MAb RU-1 reacted only with NSA-SKase. All nephritis-associated group A streptococcal strains tested reacted with MAb N-59; 87.5% of these strains reacted with MAb RU-1. MAb N-59 reacted with SKase produced by group G streptococcal strains isolated from patients with PSGN, and MAb RU-1 recognised SKase in two out of three of these strains.