Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue-type [t-PA] and urokinase-type [u-PA]) and for fibrin. In this study, anti-PAI-1 monoclonal antibodies (MoAbs) were used to identify interaction sites of PAI-1 with these components. The binding sites of 18 different MoAbs were established and are located on five distinct “linear” areas of PAI-1. MoAbs, binding to two distinct areas of PAI-1, are able to prevent the inhibition of t-PA by PAI-1. In addition, two interaction sites for fibrin were identified on PAI-1. The area located between amino acids 110 and 145 of PAI-1 contains a binding site for both components and its significance is discussed in the context of the t-PA inhibition by fibrin-bound PAI-1. Subsequently, the MoAbs were used to assess the role of platelet-PAI-1 in clot-lysis. An in vitro clot-lysis system was used to demonstrate that clot-lysis resistance is dependent on the presence of activated platelets and that PAI-1 is a major determinant for lysis-resistance. We propose that, upon activation of platelets, PAI-1 is fixed within the clot by binding to fibrin and retains its full capacity to inhibit t-PA and u-PA.
The new 10% liquid immunoglobulin preparation was well tolerated and shown to have an excellent pharmacokinetic, efficacy and safety profile. The liquid formulation provides convenience to patients and healthcare professionals.
Calcium plays a dual role in the activation of protein C: it inhibits protein C activation by a-thrombin, whereas it is required for protein C activation by the thrombomodulin-thrombin complex. Available information suggests that these calcium effects are mediated through calcium induced structural changes in protein C. In this paper, we demonstrate that substitution of Aspl67 (located in the activation peptide of human protein C, occupying position P3 relative to the peptide bond Argl69-Leul7O which is susceptible to hydrolysis by thrombin) by either Gly or Phe results in protein C derivatives which are characterized by an altered response to calcium. At 3 mM calcium, a-thrombin activated the derivatives 5-to 8-fold faster compared with the wild-type, an effect which was shown to be caused by a decreased inhibitory effect of calcium on the reaction. These same single amino acid substitutions enhanced the affinity of the thrombomodulin-thrombin complex for the substrate at 3 mM calcium 3-(Glysubstitution) to 6-(Phe-substitution) fold, either without influencing kc.t (Gly-substitution) or with a 2.5-fold decrease of keat. For both derivatives, the calcium concentrations resulting in half maximal inhibition of activation by ct-thrombin and in half maximal stimulation of activation by the thrombomodulin -thrombin complex increased from 0.3 mM to 0.6 mM. It is concluded that Aspl67 is involved in the calcium induced inhibition of protein C activation by thrombin. Moreover, our studies demonstrate that it is feasible to enhance the efficiency of enzymatic reactions by introducing point mutations in the substrate.
Inhibitor formation is currently recognized as the most serious complication of haemophilia A therapy. Development of inhibitors is generally restricted to previously untreated patients, most commonly children with severe haemophilia A, and is rarely encountered after 50 days of cumulative exposure to exogenous factor VIII (FVIII). While inhibitors of low titre usually prove to be transient and infrequently lead to serious complications, highresponder inhibitors, i.e. titres of 10 Bethesda units (BU) or greater, are associated with marked resistance to administered FVIII and major clinical challenges in managing bleeding episodes.A variety of patient-, therapy-and assay-related factors in¯uencing inhibitor formation and detection have recently been outlined [1]. Among the therapyrelated factors is the type of administered FVIII product. For example, outbreaks of inhibitors have been associated with switching previously treated patients (PTPs) to plasma-derived FVIII (pdFVIII) virally inactivated by pasteurization in conjunction with either prior controlled-pore silica adsorption [2,3] or solvent/detergent treatment [4]. These viral inactivation methods alter the properties of the FVIII molecule, resulting in exposure of neoantigens. In the absence of such molecular alterations, however, switching FVIII products does not per se appear to increase the risk of de novo inhibitor development. In an investigation of 73 patients receiving FVIII exclusively at one treatment centre, the frequency of inhibitor formation in patients who had been switched from one FVIII product to another was actually lower compared to that in recipients of a single product, with an odds ratio of 0.4 and 95% con®dence interval (95% CI) of 0.1±2.1, although this difference was not statistically signi®cant [5].Recombinant FVIII (rFVIII) has been increasingly adopted in the therapeutic management of haemophilia A. This form of FVIII offers a very high degree of viral safety, as well as increased availability because it is not dependent upon human plasma supplies. rFVIII has proven to be no more immunogenic than other FVIII products when account is taken of all factors known to affect inhibitor formation and detection [6,7].Nevertheless, some published data might suggest an increased risk of inhibitor development in PTPs switched from pdFVIII to rFVIII. In one study [8], for instance, 62 multitransfused haemophilia patients were followed for 3 years. During the eighth month of the study, ®ve patients were switched to rFVIII. Three of the ®ve developed de novo inhibitors. However, the peak inhibitor titre in all three cases was £ 1 BU, and there were no associated complications. A case report has also appeared of lowresponder inhibitor formation (2.1 BU antihuman and 1.3 BU antiporcine) in a PTP exposed to two Summary. De novo inhibitor development is a rare event in PTPs switched from pdFVIII to rFVIII. Based on previously published data of clinical studies a change in FVIII product is unlikely to provoke inhibitor formation.
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