Effect of phenylglyoxal-modified alpha2-antiplasmin on urokinase-induced fibrinolysis.

One of the functions of activated blood clotting factor XIII (FXIIIa) is the crosslinking of alpha2-antiplasmin (alpha2AP) to fibrin. This process results in localization and concentration of alpha2AP throughout fibrin, thereby making fibrin more resistant to digestion by plasmin. We reasoned that competition by chemically-modified inactive alpha2AP (mod alpha2AP) with native alpha2AP would diminish the resistance of fibrin to digestion by plasmin. Mod alpha2AP was prepared by treating native alpha2AP with an Arg-specific reagent, phenylglyoxal. An average of four of the total nineteen Arg residues in alpha2AP reacted with phenylglyoxal and resulted in complete loss of plasmin inhibitory activity; however, mod alpha2AP competed effectively with native alpha2AP for becoming crosslinked to fibrin by FXIIIa catalysis. In the presence of mod alpha2AP, urokinase (UK)-induced plasma clot lysis time shortened significantly. Mod alpha2AP enhanced UK-induced clot lysis in a whole blood system as shown by the similarities of rates of clot lysis for a mixture of 20 U/ml UK and 1.5 microM mod alpha2AP versus that induced by 100 U/ml UK without mod alpha2AP. Less fibrinogenolysis occurred in whole blood when mod alpha2AP was present since much lower UK concentrations were needed to achieve the same level of fibrinolysis than when only native alpha2AP was present. Our results indicate that mod alpha2AP enhances UK-induced fibrinolysis by competitive inhibition of factor XIIIa-mediated incorporation of native alpha2AP into fibrin.

Purification of human alpha 2-antiplasmin with chicken IgY specific to its carboxy-terminal peptide.

alpha 2-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to alpha 2-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and alpha 2-antiplasmin, pure alpha 2-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of alpha 2-antiplasmin activity; and (b) the alpha 2-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the alpha 2-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.

RGDFAP: platelet aggregation inhibitory and profibrinolytic hybrid peptide (RGDF coupled with the carboxy terminal part of alpha 2-antiplasmin) enhances plasminogen binding to platelets.

As published in a recent issue of Blood Coagulation and Fibrinolysis, the hybrid peptide RGDFAP, composed of RGDF (Arg-Gly-Asp-Phe) coupled to a synthetic peptide residue of the carboxy terminal part of antiplasmin (AP26) inhibited platelet activation and augmented plasmin generation and in vitro fibrin clot lysis. This peptide contains an RGD motif which provides linkage to platelet GP IIb-IIIa. The antiplasmin part of the molecule may attach free plasminogen, which in turn increases the amount of platelet surface bound plasminogen, probably yielding enhanced lytic action at the site of thrombus formation. This hypothesis was investigated and confirmed by the results of platelet-plasminogen binding assays, using FITC-labelled antiplasmin antibodies and radioligand binding analysis. Increased platelet-linked plasminogen was detected by a chromogenic method, along with the acceleration of in vitro lysis of platelet-rich clots in the presence of RGDFAP peptide.

Hybrid peptide containing RGDF (Arg-Gly-Asp-Phe) coupled with the carboxy terminal part of alpha 2-antiplasmin capable of inhibiting platelet aggregation and promoting fibrinolysis.

The aim of this study was to synthesize and investigate hybrid peptides which contain the RGD (Arg-Gly-Asp) sequence coupled with lysine residues in special arrangements (antiplasmin carboxyterminal peptide) in an effort to simultaneously inhibit platelet aggregation and promote fibrinolysis. The in vitro haemostatic modifying properties of the synthesized peptides were tested by ADP-induced platelet aggregation, plasmin-generation tests and fibrin-clot lysis assays. The hybrid peptide RGDFAP, composed of RGDF (Arg-Gly-Asp-Phe) coupled to a synthetic peptide residue of the carboxyterminal part of antiplasmin (AP26) inhibited platelet activation and increased plasmin generation and in vitro fibrin-clot lysis.

[A new thrombocyte aggregation-inhibiting and fibrinolysis-promoting synthetic molecule: RGDF (Arg-Gly-Asp-Phe) coupled with the carboxyterminal antiplasmin peptide]. / A thrombocyta-aggregatiót gátló, fibrinolysist fokozó új szintetikus molekula: antiplasmin carboxyterminális részéhez kapcsolt RGDF (Arg-Gly-Asp-Phe).

OBJECTIVE: Synthesize such hybrid peptides, which contain the RGD (Arg-Gly-Asp) sequence coupled with another peptide containing lysine residues in special positions, to inhibit simultaneously platelet activation and promote fibrinolytic processes. DESIGN: The in vitro haemostasis modifying properties of the synthesized peptides were tested with ADP induced platelet aggregation, in vitro plasmin generation tests and fibrin-clot lysis assays. RESULTS AND CONCLUSIONS: RGDF (Arg-Gly-Asp-Phe) coupled with the carboxyterminal antiplasmin peptide (RGDFAP hybrid molecule) has a common concentration range for inhibiting platelet activation and increase plasmin generation along with accelerated in vitro fibrin-clot lysis.

Characterization and cross-reactivity of rabbit antisera to plaque toxins.

The effects of heat labile, high molecular weight water-soluble toxins from bacterial plaque on HL60 promyelocytic cells were examined. On gel filtration, four inhibitors of HL60 cell growth and two inhibitors of HeLa cell growth (PT1, PT2) were detected. The first and third HL60 cell inhibitors corresponded to the two HeLa cell inhibitors. The last eluted HL60 cell inhibitor (plaque leukotoxin, PL) did not inhibit HeLa cell growth. Anti-PT2 antibodies reduced the activity of enriched PT2 by 20-50%, but all other antisera tested exhibited no effect. Anti-PL antibodies detected antigens from Actinobacillus actinomycetemcomitans, although anti-A. actinomycetemcomitans and anti-Capnocytophaga sputigena antibodies did not react with plaque extract. These findings suggest that the plaque toxins examined in this study were probably not derived from these two bacteria.