Human Alpha2-antiplasmin Research Articles

Abstract 504: Circulating Alpha-2-Antiplasmin Affects Microvascular Thrombosis, Ischemic Neurodegeneration and Mortality in Mice Following Cerebral Thromboembolism

Introduction: For unknown reasons, clinical studies suggest that high circulating levels of human alpha2-antiplasmin may increase the risk of thrombotic vascular diseases such as stroke and contribute to the failure of tissue plasminogen activator therapy. Hypothesis: We tested the hypothesis that circulating alpha2-antiplasmin affects thrombosis, tissue injury and fibrinolysis in a translational model of middle cerebral artery thromboembolism. Methods and Results: Elevated alpha2-antiplasmin levels significantly decreased thrombus dissolution (p<0.001), enhanced matrix metalloproteinase-9 (MMP-9) expression (p<0.001) and worsened brain injury (p<0.001) and swelling (p<0.001). Conversely, genetic deficiency of circulating alpha2-antiplasmin, enhanced dissolution of thromboemboli (p<0.001) and reduced brain injury (p<0.001). Circulating alpha2-antiplasmin had dynamic effects on stroke outcomes because specific alpha2-antiplasmin inactivation after thromboembolism saved lives (p<0.001), prevented disability, decreased brain infarction (p<0.001), reduced hemorrhage (p<0.05) and brain swelling (p<0.001). Inactivation of alpha2-antiplasmin led to reductions in MMP-9 (p<0.001), microvascular thrombosis (p<0.001), blood brain barrier breakdown and apoptosis (p<0.01). Conclusions: These data suggest that alpha2-antiplasmin has deleterious effects in experimental stroke, which are mediated through the fibrinolytic pathway to alter culprit thrombus dissolution, as well as through other mechanisms that contribute to downstream microvascular thrombosis, hemorrhage, MMP-9 expression, apoptosis and blood brain barrier leakage.

Matrix metalloproteinases and cellular fibrinolytic activity.

Several molecular interactions between the matrix metalloproteinase (MMP) and the plasminogen/plasmin (fibrinolytic) system may affect cellular fibrinolysis. MMP-3 (stromelysin-1) specifically hydrolyzes urokinase (u-PA), yielding a 17 kD NH2-terminal fragment containing the functionally intact receptor (u-PAR)-binding sequence and a 32 kD COOH-terminal fragment containing the intact serine proteinase domain. MMP-3 generates an angiostatin-like fragment (containing kringles 1-4 with the cellular binding domains) from plasminogen. Treatment with MMP-3 of monocytoid THP-1 cells saturated with bound plasminogen, resulted in a dose-dependent reduction of the amount of u-PA-activatible plasminogen. Treatment with MMP-3 of cell-bound u-PA, in contrast, did not alter cell-associated u-PA activity. These data thus indicate that MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, without affecting cell-bound u-PA activity. MMP-3 also specifically interacts with the main inhibitors of the fibrinolytic system. Thus, MMP-3 specifically hydrolyzes human alpha2-antiplasmin (alpha2-AP), the main physiological plasmin inhibitor. alpha2-AP cleaved by MMP-3 no longer forms a stable complex with plasmin and no longer interacts with plasminogen. Cleavage and inactivation of alpha2-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis. Furthermore, MMP-3 specifically hydrolyzes and inactivates human plasminogen activator inhibitor-1 (PAI-1). Stable PAI-1 bound to vitronectin is cleaved and inactivated by MMP-3 in a comparable manner as free PAI-1; the cleaved protein, however, does not bind to vitronectin. Cleavage and inactivation of PAI-1 by MMP-3 may thus constitute a mechanism decreasing the antiproteolytic activity of PAI-1 and impairing the potential inhibitory effect of vitronectin-bound PAI-1 on cell adhesion and/or migration. These molecular interactions of MMP-3 with enzymes, substrates and inhibitors of the fibrinolytic system may thus play a role in the regulation of (cellular) fibrinolysis. Furthermore, the temporal and topographic expression pattern of MMP components, as well as studies in gene-deficient mice, suggest a functional role in neointima formation after vascular injury.

Enzymatic properties of phage-displayed fragments of human plasminogen.

Two low-molecular-mass forms of human plasminogen, plasminogen-(543-791)-peptide (micro-plasminogen), comprising the serine protease domain, and plasminogen-(444-791)-peptide (mini-plasminogen), which in addition contains kringle 5, were displayed on filamentous phage by fusion to the N-terminus of the minor coat protein pIII, to levels of 0.5 molecules micro-plasminogen-pIII/phage particle and 0.1 molecules mini-plasminogen-pIII/phage particle. The proenzymes, quantitatively activated by urokinase, showed catalytic efficiencies that were virtually identical to their soluble counterparts, and activity remained associated with the phage as demonstrated by phage ELISA and biopanning with human alpha2-antiplasmin or the inhibitor Phe-Pro-Arg-CH2Cl. Micro-plasminogen-pIII was activated by streptokinase and staphylokinase, two non-enzymatic plasminogen activators, to the same extent as by urokinase. Activated forms of mini-plasminogen-pIII micro-plasminogen-pIII and mini-plasminogen dissolved 125I-labelled fibrin films in a dose-dependent time-dependent manner, with 50% lysis in 20 h requiring 0.52, 3.2 and 0.46 nM active plasmin, respectively. Thus, proenzyme moieties derived from plasminogen can be successfully displayed on phage with maintenance of their enzymatic properties. The micro-plasminogen and mini-plasminogen phage-display systems may be useful to study mechanisms of plasminogen activation.

Inhibition of clot-bound alpha 2-antiplasmin enhances in vivo thrombolysis.

Recent experiments in vitro have shown that inhibition of human alpha 2-antiplasmin by a monoclonal antibody (MAb RWR) markedly enhances clot lysis by plasminogen activators. To extend these studies in vivo, we tested whether inhibition of clot or fibrin-bound alpha 2-antiplasmin by MAb RWR could enhance the lysis of a human clot by tissue-type plasminogen activator (t-PA) in a rabbit jugular vein thrombosis model. Compared with a saline placebo or a control antibody, MAb RWR significantly increased thrombolysis by endogenous plasminogen activator in rabbits to which no t-PA was administered (p less than 0.05). In rabbits that received t-PA, the combination of MAb RWR and t-PA caused significantly greater thrombolysis than equivalent doses of t-PA alone (p less than 0.05). However, compared with equipotent doses of t-PA alone, the combination of MAb RWR and t-PA did not increase the nonspecific consumption of fibrinogen. These experiments suggest that the combination of an alpha 2-antiplasmin inhibitor and a plasminogen activator could be a more potent thrombolytic strategy.

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin).

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.

Modification of a tyrosine residue in the carboxyterminal portion of human alpha 2-antiplasmin

Alpha*-antiplasmin, the main physiological plasmin inhibitor in human plasma, is a single chain glycoprotein with a molecular weight of 70,000, containing about 13 percent carbohydrate, about 500 amino acids and 3 disulfide bridges (1). antithrombin III, Alpha2-antiplasmin belongs to the same protein family as alpha d-antitrypsin and ovalbumin (2). Its COOH-terminal amino acid sequence was etermined and shown to extend at least 52 amino acids beyond the COOH-terminal end of antithrombin III (3). During the reaction between plasmin and alpha -antiplasmin a stable complex is formed following a plasmic attack at a specs ic Arg-Met peptide bond situated about 80 residues + from the C-terminus in alpha2-antiplasmin. This peptide bond is cleaved in the native complex indicating that most likely an ester bond is stabilizing the complex (4,5).

Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human alpha 2-antiplasmin.

23 hybridomas secreting monoclonal antibodies against human alpha 2-antiplasmin, the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class IgG2b. All light chains belong to the kappa group. The specificity and relative avidity of these monoclonals have been determined using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for alpha 2-antiplasmin, 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 monoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the alpha 2-antiplasmin molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.