Presence Of Fibrinogen Fragments Research Articles

A New Role for Marine Skeletal Proteins in Regenerative Orthopaedics

Use of ready-made marine skeletons is one of the simplest possible remedies to major problems hindering the future development of regenerative orthopaedics- such as, providing a richness of framework designs and now a potentially rich, accessible source of osteopromotive analogues and biomineralisation proteins. It has already been shown that coral and marine sponge skeletons can support self-sustaining musculoskeletal tissues and that extracts of spongin collagen and nacre seashell organic matrices promote bone mineralisation. This should not be surprising given that the pivotal biomineralisation proteins, which orchestrate bone morphogenesis are also found in the earliest calcifying marine organisms. This is because they are representatives of the first molecular components established for calcification, morphogenesis and wound healing. In support of this notion, it has emerged that BMP molecules- the main cluster of bone growth factors for human bone morphogenesis- are secreted by endodermal cells into the developing skeleton. In addition, the regenerative signalling proteins, TGF-b and Wnt-prime targets in bone therapeutics- are also present in early marine sponge development and instrumental to stem cell activation in Cnidarians. Based on this match between vertebrate and invertebrate main developmental proteins, we review the nature and extent of this evolutionary relatedness and use it to support the development of a new strategy, which is to mine selected marine origin organic matrices for novel metabolic, signalling and structural proteins/peptides and protein analogues to apply in regenerative orthopaedics, particularly when using adult stem cells. To support such a proposal we show early stage evidence-gathered in our own laboratory- of the presence of fibrinogen fragments and early osteopromotive effects of a coral organic matrix extract on stem cells. In practice the discovery of new osteopromotive and osteo-accelerant protein analogues will require use of traditional chromatography techniques, osteoactivity assays to hone in on potential proteins of significance and advanced proteomic tools to provide accurate sequencing, determine the mechanisms and molecular pathways involved in osteoactivation and determine the efficiency and effectiveness of marine skeleton-derived protein modulation of the stem cell (MSC) proteome. As more analogues are discovered using proteomic tools, skeletal organic matrices may have ever increasing utility for regenerative orthopaedics.

Comparison of the inhibition of urokinase-type plasminogen activator (u-PA) activity by monoclonal antibodies specific for u-PA as assessed by different assays

Six murine monoclonal antibodies (MAbs) specific for urokinase-type plasminogen activator (u-PA) were tested for their ability to inhibit u-PA activity in three different assays with respect to amidolytic activity, plasminogen activation and fibrinolytic activity. Two of the MAbs were able to inhibit the amidolytic activity of u-PA. One MAb (LMW 3.3) inhibited at low concentrations, but only partially, whereas high concentrations of MAb LMW 2.3 could almost completely inhibit the u-PA activity. Testing the MAbs in the u-PA-mediated plasminogen activation assay, both in the presence and absence of fibrinogen fragments, and in the fibrinolytic assay, resulted in different inhibition patterns. The u-PA-mediated plasminogen activation in the presence of fibrinogen fragments was inhibited by high concentrations of all six MAbs. However, in the absence of fibrinogen fragments only MAb LMW 3.3 partially blocked the u-PA activity. In the u-PA-mediated fibrinolytic assay, besides MAb LMW 3.3, the kringle specific MAbs UK 2.1 and UK 50.2 were also able to inhibit the u-PA activity. The results indicate that the inhibition patterns of the MAbs can differ dramatically between different assays. In addition, the concentrations of the MAbs used in the assays can be very important as is illustrated by MAb UK 50.2, which showed in some assays a stimulating as well as an inhibitory effect dependent on the concentration of the MAb.

A comparison of cultured cells with other promoters of tissue plasminogen activator kinetics

Summary Single chain tissue plasminogen activator (sctPA) is not a zymogen form of two chain tPA (tctPA) but its activity can be enhanced by this conversion (by plasmin) and also in the presence of various promoter species, e.g. fibrin, fibrinogen fragments, heparin. Promoter stimulation is usually found to be more effective than proteolytic activation of tPA and this has been confirmed here using time courses from sc- to tctPA reactions in plasminogen activation assays with a wide range of promoters under similar conditions. Promoters investigated were fibrinogen fragments (FF), soluble fibrin (DESAFIB), unfractionated heparin (UFH), low molecular weight heparin (LMWH) and solid fibrin for comparison with cell surfaces from a number of transformed cell lines grown in culture. Sc- to tctPA conversion gave up to 4-fold improvements in plasminogen activation rates, much less than the stimulation due to promoters, up to 77-fold. The best promoters were FF and cell surfaces. Promoters were active on both sc- and tctPA. More forcing plasmin treatment of sctPA produced a further degradation product of tctPA, termed low molecular weight tPA (LMW tPA). This product has lost the finger domain (amino acids 1–49) and has a significantly different activity profile from full length tPA. LMW tPA maintains activity with chromogenic substrates and in the presence of fibrin and DESAFIB, but activity is reduced close to unstimulated levels in the presence of FF, heparin and cell surfaces. Thus, two mechanistic groups of promoters emerge: (1) FF, heparin and cell surface receptors and (2) DESAFIB and fibrin, where tPA finger-promoter interactions are essential for group 1, partially involved in group 2.

The Activation of Type 1 and Type 2 Plasminogen by Type I and Type II Tissue Plasminogen Activator

Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.

Characterization of a tissue-type plasminogen activator from porcine urine

Porcine urine, unlike human urine, does not contain detectable amounts of urokinase-type plasminogen activator (u-PA). The plasminogen activator present in porcine urine is of tissue-type (t-PA) as identified by the following criteria. (1) Porcine urine PA exhibits an M r of 65 000 similar to the M r of human t-PA (64-70 000) but distinct from the M r of human u-PA (55 000). (2) Antibodies against human t-PA bind and inhibit crude and purified porcine urine PA, while human u-PA-specific antibodies do not react with porcine urine PA. (3) Plasminogen activation by porcine urine PA is markedly stimulated in the presence of fibrinogen fragments. (4) Porcine urine PA activity is not affected by concentration of amiloride substantially suppressing human u-PA activity.

Partial Purification and Characterization of Native Plasminogen Activators from Bovine Milk

At least four native plasminogen activators were detected in bovine milk, and two partially purified plasminogen activators were characterized. The plasminogen activators were dissociated from casein proteins by treatments with sulfuric acid and dimethylformamide. The plasminogen activators in the resulting fractions were partially purified with size exclusion, affinity, or metal chelate chromatographic techniques. Molecular weights of the two partially purified plasminogen activators were 47.2 and 30.5 kDa by gel electrophoresis. Size exclusion chromatography gave a molecular weight of 43.2 kDa for the first plasminogen activator. The isoelectric points of the two plasminogen activators were in the pH range 6.2 to 6.7. Because activity was not enhanced by the presence of fibrinogen fragments in a plasminogen activator assay mixture and decreased when human anti-urokinase Ig were added, at least some bovine milk native plasminogen activators appear to be urokinase-type plasminogen activators.

Plasminogen activation in diabetes mellitus. Kinetic analysis of plasmin formation using components isolated from the plasma of diabetic donors.

Two components of the fibrinolytic system, plasminogen and the vascular plasminogen activator, have been isolated to apparent homogeneity from the post-venous occlusion plasma of three diabetic patients (hemoglobin A1C greater than 7%) and of one nondiabetic control person. Plasminogen activation was studied for each person separately in the absence and presence of CNBr fragments of fibrinogen. Activation of diabetic plasminogen by urokinase was not significantly altered as compared to the activation of control plasminogen. The same was found when diabetic plasminogen was activated by control vascular plasminogen activator in the presence of fibrinogen fragments but only at plasminogen concentrations below 10-30 nM; at higher substrate concentrations, however, plasminogen activation was impaired in a pattern resembling substrate inhibition. Activation of control plasminogen by diabetic vascular plasminogen activator was completely impaired in the absence of fibrinogen fragments. Addition of fibrinogen fragments stimulated plasmin formation by diabetic vascular plasminogen activator resulting in kinetic constants which were similar to the activation of control plasminogen by control vascular plasminogen activator in the absence of fibrinogen fragments (Km = 7.5 microM, kcat = 0.05 S-1). Addition of fibrinogen fragments in controls decreased Km values to less than 0.1 microM. Despite addition of fibrinogen fragments the rate of plasmin formation from diabetic plasminogen by diabetic vascular plasminogen activator isolated from the same diabetic donor was so small that kinetic constants could not be calculated.