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Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character, and the P-2 and P-3 substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.

Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.

The fibrinolytic system is involved in a wide variety of biological phenomena such as dissolution of fibrin blood clots, tissue remodeling, metastasis, angiogenesis, wound healing, embryogenesis and embryo implantation (1). It differs physiologically in neonates from older children or adults. The absolute and relative concentrations of various components differ from adults, and they are dependent on both the gestational age and postnatal age (for review see 2). In addition, the functional behavior of fetal plasminogen and fetal fibrinogen differ from the adult forms, although electrophoretic molecular weight analyses, as well as amino acid composition and partial amino acid sequencing revealed no differences between fibrinogen and between the plasminogen forms 1 and 2 of neonates and adults (3–12). Therefore, variations in functional behavior have been related to differences in carbohydrate composition (5). Oligosaccharides linked to proteins may contribute to receptor-mediated interactions, protein stability, clearance from the circulation, and physiological function (13,14).

The generation of active two chain urokinase from the proenzyme pro-urokinase by catalytic amounts of plasmin is an important regulatory step in plasminogen activation. The plasminogen activator, urokinase, converts plasminogen to plasmin which in turn dissolves fibrin blood clots. We have studied the conversion of pro-urokinase (Pro-UK) to high molecular weight urokinase (HMW-UK) by plasmin. This paper describes a peptide map that separates various plasmin cleavage products of Pro-UK/HMW-UK. A partially purified sample of HMW-UK containing trace amounts of plasmin was incubated at room temperature for 12 days. The digested peptide mixture after reduction and S-carbamide methylation was separated using reversed phase high performance liquid chromatography. The peptides were collected and analyzed for their N-terminal amino acid sequences, and in some cases by plasma desorption mass spectrometry (PDMS). The plasmin map so generated is useful in investigating the integrity of any HMW-UK produced via plasmin cleavage step.

Scleral implants are made of absorbable or nonabsorbable materials. One category of absorbable materials consists of donor tissue, either autogenous (fascia lata, plantaris gracilis tendon) or from cadavers (dura mater, sclera, fascia lata). A second category includes gelatin, reconstituted collagen, absorbable gut, fibrin, blood plasma, air, and sodium hyaluronate; of these, specially prepared gelatin seems to be the most useful. Nonabsorbable implant materials have proven more practical than absorbable implants. Solid silicone rubber is currently the most popular scleral implant material. It is soft, easy to handle, and well tolerated. Expandable implants, either temporary or permanent, are used in the form of a silicone balloon filled with liquid. Silicone sponge, also widely used, has the advantage of great softness. However, hydrogels seem to be the ideal scleral implants. The only one commercially available is Refojo's MAI implant. It is very soft and is not damaged by sutures, has a smooth surface, is molded in several sizes and shapes, and has small pores that microorganisms cannot penetrate. When saturated with a water-soluble antibiotic before implantation, this implant releases the antibiotic postoperatively for a longer time than any other implant material. Finally, surgical adhesives are useful when the sclera is too thin or weak to tolerate sutures. The best adheive available seems to be isobutyl cyanoacrylate.

Apolipoprotein B (apo B), fibrinogen/fibrin, blood platelets, factor VIII-related antigen of the blood coagulation system, and smooth muscle cells (SMC) were identified in the intima of normal and atherosclerotic human aorta and large arteries by the indirect immunofluorescence technique. Fibrinogen/fibrin was revealed by a monoclonal antibody (monAb) against the C-terminal region of human fibrinogen A a-chain. Fibronectin was visualized by monAb to the cellular form and against an epitope shared by different fibronectin subunit variants. In normal intima, fatty streaks, small amounts of fibrinogen/fibrin together with large amounts of apo B were observed. Fibronectin detected by two types of monAb was not found in extracellular matrix (ECM), whereas cellular fibronectin encircled SMC. According to the data obtained, fibrinogen/fibrin accumulates in plaques as a result of intramural thrombus incorporation, blood insudation, intramural haemorrhage, and in or around cells, apparently macrophages.

In order to assess systemic and local intra-arterial levels of D-dieter, a specific degradation product of cross-linked fibrin, blood samples were obtained before and 0.5, 1.5, 4, 8, 24, and 48 hours after the start of intra-arterial low dose streptokinase treatment (3750 IU/h) in 21 patients with angiographically documented peripheral arterial occlusive disease. Blood was sampled simultaneously from the cubital vein and from the arterial catheter located close to the occlusive thrombus. Pre-treatment D-dieter levels in arterial and venous blood were 298 and 259 ng/mL (medians), respectively. During the first day of treatment D-dieter levels in arterial blood increased on average 10–20 fold and were at all times significantly higher than D-dieter levels in venous blood (artery vs vein; 0.5 h: 414 vs 266; 1.5 h: 2640 vs 411; 4 h: 2490 vs 898; 8 h: 2640 vs 906; 24 h: 6290 vs 1060 ng/mL, medians). In 13 patients in whom recanalization was achieved D-dieter levels were higher than in 8 unsuccessfully treated patients (vein: p < 0.05; artery: not significant). Significant decreases in plasminogen, α2-antiplasmin, fibrinogen and euglobulin clot lysis time indicated fibrinolytic activation in local arterial as well as in systemic venous blood. It was concluded that higher D-dieter in the vicinity of the thrombus compared to systemic levels reflected local lysis of fibrin. Systemic levels of D-dieter might provide early information on the efficacy of the treatment.

Fluorescent basic drugs have been shown to selectively accumulate in granules of blood platelets. The transformation kinetics of these organelles was studied in the course of clot formation. Simultaneous recording of fluorescence intensity and light transmission demonstrated that processes of fibrin filaments sedimentation and fibrin-blood platelet interaction were followed by the “collapse” of cells and liberation of the fluorescent marker. The platelet degranulation takes place due to contractile properties of platelets. This process was found to be inhibited by cytochalasin B and colchicine. Redistribution of platelets into aggregated, caused by the addition of ADP and stirring, led to the inhibition of the liberation. These experiments provide the evidence that secretion by platelets is realized by different mechanisms. Fluorescent basic drugs are a useful tool for investigating the function of the granules of blood platelets.

Summary Subacute bacterial endocarditis is a distinet pathologic and clinical entity. The vegetative endocarditis that characterizes this disease, with its exudation of fibrin, blood platelets, and enmeshed bacteria, represents a state of high local and general tissue immunity to bacterial invasion, rather than lowered resistance; the immunity resides in the local endothelial structures, as well as in the general reticuloendothelial system. Since the reaction to this bacterial invasion is largely endothelial, the disease can rightfully be termed infectious endotheliosis . Subacute bacterial endocarditis occurs typically in patients who have valvular disease, usually rheumatic, but who have acquired a high degree of immunity, so that reinfection, recurrent endocarditis or pancarditis, and congestive heart failure do not occur. This increased immunity is responsible for the fact that the patient is not heart-conscious even during the attack of acute valvular infection. Then a transient bacteremia, caused by an upper respiratory infection, tonsillectomy, the extraction of an infected tooth, or pyelitis, permits secondary invasion and implantation of bacteria (usually the ubiquitous Streptococcus viridans ) on the damaged valve. A careful history will reveal that some infection which lowered the general body resistance is usually the beginning of an illness that later develops into subacute bacterial endocarditis. Although the bacteria are able to implant themselves on the damaged valves, they find in the valve an altered tissue reactivity which does not permit much local damage, such as ulceration or extension into the myocardium or pericardium. A more favorable outcome would be likely if the bacteria were not localized in a focus that communicates directly with the blood stream. If embolism does not cut the disease short, death occurs when the local and systemic endothelial systems become exhausted. Subacute bacterial endocarditis is a true endocardial sepsis, for the valve acts as the primary distributing focus of the infection. As such, if the focus can be removed surgically (as in some cases of patency of the ductus arteriosus) or chemotherapeutically before complications set in, there is some hope for a cure of this dreaded disease.