The interactions between antithrombin III,thrombin and surface immobilized heparin

The interactions between antithrombin III (ATIII), thrombin, and surface immobilized heparin were investigated. Carboxylated polystyrene modified with covalently immobilized albumin-heparin conjugate contain sites which can bind ATIII from buffer and plasma solutions. Approximately 65% of the ATIII molecules present at the heparinized surface either adsorbed from a buffer or plasma solution, exchanged with ATIII in buffer solution. The exchange between surface bound ATIII and ATIII in solution was repeated several times on the same heparinized surface. The number of binding sites that could bind and release ATIII was much higher when the heparinized surface was exposed to an ATIII containing buffer solution than to a plasma solution. The reduction in binding sites available for ATIII using plasma solutions as compared to buffer solutions could be explained by the competition of other plasma proteins with ATIII for the heparinized surface. It was observed that heparin binding proteins were able to compete with ATIII for binding to the immobilized heparin. Furthermore adsorption of proteins on the heparinized surface significantly reduced the availability of binding sites for ATIII. Exposure of thrombin to the heparinized surface resulted in thrombin activity at the surface. The thrombin activity on the heparinized surfaces was lower on surfaces with a higher ATIII concentration. The activity of surface bound thrombin was not affected by the presence of other plasma proteins. Enzymatically active thrombin molecules present at the heparinized surface were completely inactivated when the surface was exposed to a solution containing ATIII. The inactivation rate of surface bound thrombin by ATIII was higher than the rate of the uncatalyzed inactivation of thrombin in solution. Part of the Thrombin-Antithrombin III (TAT) complexes (10-20%) that were formed upon inactivation of thrombin remained bound to the heparinized surface. In general it was concluded that only the surface immobilized heparin molecules that can bind ATIII in a reversible way determine the anticoagulant properties of the surface. The mechanism of inactivation of a protease on a heparinized surface depends either on the catalytic effect of heparin on the inactivation rate of proteases by ATIII or on an increased uncatalytic inactivation due to increased concentrations of ATIII near the surface as compared to the concentration of ATIII in the bulk phase.     

Interactions of biospecific functional polymers with blood proteins and cells

Biospecific functional polymers, i.e. synthetic or artificial polymers substituted with specific chemical functional groups carried by the macromolecular chain are designed to interact with living systems. These polymers are either insoluble or soluble, derived from polystyrene and dextran. Polymers substituted with aryl sulfonate and carboxyl groups specifically interact with antithrombin III and serine-proteases involved in the coagulation of blood. As a consequence, these polymers possess heparin-like activity and are therefore of low thrombogenicity when exposed to flowing blood. Other functional polymers have been prepared in order to interact with various components of the immune system. Soluble and insoluble functional polymers in contact with cells can affect both cell proliferation and metabolism. Some functional polymers have the ability to inhibit or to stimulate cell growth while others can alter cell function without a change in growth characteristics. The functional polymers described have possible applications as plasma expanders, non-thrombogenic catheters, non-complement activating surfaces and other applications in oncology, biotechnology and immunochemistry.     

Interactions of biospecific functional polymers with blood proteins and cells

Biospecific functional polymers, i.e. synthetic or artificial polymers substituted with specific chemical functional groups carried by the macromolecular chain are designed to interact with living systems. These polymers are either insoluble or soluble, derived from polystyrene and dextran. Polymers substituted with aryl sulfonate and carboxyl groups specifically interact with antithrombin III and serine-proteases involved in the coagulation of blood. As a consequence, these polymers possess heparin-like activity and are therefore of low thrombogenicity when exposed to flowing blood. Other functional polymers have been prepared in order to interact with various components of the immune system. Soluble and insoluble functional polymers in contact with cells can affect both cell proliferation and metabolism. Some functional polymers have the ability to inhibit or to stimulate cell growth while others can alter cell function without a change in growth characteristics. The functional polymers described have possible applications as plasma expanders, non-thrombogenic catheters, non-complement activating surfaces and other applications in oncology, biotechnology and immunochemistry.     

Interactions of proteins in human plasma with modified polystyrene resins

Investigations are reported on the composition of protein layers adsorbed from plasma to various modified polystyrene resins. As well as polystyrene itself, polystyrene bearing sulfonate groups in the benzene rings, and polystyrene sulfonate in which the sulfonate groups were converted to amino acid sulfamide, were investigated. Some of these resins were shown in previous work to have anticoagulant properties. To study the adsorption of proteins from plasma, the resins were exposed to citrate anticoagulated human plasma for 3 h. Adsorbed proteins were then eluted sequentially by 1M Tris buffer and 4% SDS solution, and examined by SDS-PAGE. The gel patterns were similar on all resins except polystyrene. From the MWs of the gel bands, the major protein component appeared to be fibrinogen. Smaller amounts of plasminogen, transferrin, albumin, and IgG were also present. In addition, Ouchterlony immunoassay of the eluates from one resin gave positive identification of complement C3, fibronectin, IgG, and IgM. Many other minor gel bands remain unidentified. A consistent finding for all resins was the presence of plasmin-type fibrinogen degradation products though the amounts varied with resin type. It is concluded from this (and from experiments showing FDP formation when fibrinogen was absorbed to the resins, from buffer containing a trace of plasminogen) that the functional groups in these materials promote the adsorption of plasminogen and its activation to a plasmin-like molecule. It appears from the substantial quantities of fibrinogen adsorbed to these materials after 3 h exposure to plasma that the Vroman effect (giving transient adsorption of fibrinogen) is not operative on these materials. It is hypothesized that specific interactions occur between fibrinogen and sulfonate groups.     

Interactions of fibrinolytic system proteins with lysine-containing surfaces

Studies on the interactions of tissue plasminogen activator (tPA) and plasminogen with polyurethane surfaces containing epsilon-lysine moieties (epsilon-amino group free) are reported. These surfaces are considered to have the potential to dissolve nascent clots that may be formed on them. For adsorption from both single protein solutions and plasma, the surfaces were found to have a high capacity for tPA as well as plasminogen. A significant fraction of preadsorbed tPA was displaced from the epsilon-lysine surfaces upon contact with plasma. These surfaces, when preadsorbed with tPA and then incubated with plasma, were able to dissolve incipient clots formed around them. However, the clot-dissolving capacity diminished as the time of plasma incubation increased, presumably due to loss of tPA. It was also shown that in plasma, preadsorbed tPA is displaced from these surfaces largely by plasminogen, which thus appears to have a greater binding affinity than tPA for the epsilon-lysine moieties. Finally, it was found that in plasma, the epsilon-lysine surfaces interact with plasminogen in a dynamic manner, and that about 70% of the bound plasminogen is exchanging continuously with plasminogen in the plasma.     

Interactions of histidine-rich glycoprotein with immunoglobulins and proteins of the complement system

This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.     

Interactions of thermally denatured fibrinogen on polyethylene with plasma proteins and platelets.

During the investigation of fibrin deposition onto hydrophobic polymers in contact with human blood, a model was developed in which fibrinogen was denatured and irreversibly coated onto a polyethylene surface by heating to 70 degrees C for 10 min. The denatured fibrinogen-polyethylene surface is resistant to fluid wall shear rates of up to 550 s-1 and the fibrinogen does not desorb in the presence of plasma proteins. Compared to uncoated polyethylene, little albumin or fibrinogen adsorbs to heat-denatured fibrinogen. Thrombin binds to the denatured fibrinogen-coated polyethylene with low affinity and also acts on the surface-bound denatured fibrinogen and cleaves fibrinopeptide A (FPA) quantitatively. Washed, 51Cr-labeled platelets do not adhere to the thermally denatured fibrinogen at either low or high shear rates compared to surfaces coated with undenatured fibrinogen (p < 0.01). These observations support the role of the D domain of fibrinogen in platelet adhesion because this is the region that is denatured by heating. In contrast, the E domain of fibrinogen is not altered by heating to 70 degrees C and hence remains susceptible to thrombin and/or plasmin cleavage. The characteristics of this surface are such that it can be used to develop fibrin-coated surfaces for use in studies of thrombus formation on artificial surfaces.     

The so-called "contact" system of plasma: consequences of the activation of the Hageman factor

The contact system of plasma includes 4 zymogens, Hageman factor, pre-kallikrein, factor XI and plasminogen, and a cofactor, the high molecular weight kininogen (HMWK). The activation of the contact system leads to the production of bradykinin, blood coagulation, fibrinolysis, complement activation and neutrophil stimulation. Consequently these phenomena generate a lot of pro-inflammatory factors which contribute to the local and systemic inflammatory processes.     

Antithrombin and heparin antithrombin patterns in pre-thrombosis and thrombosis.

An antithrombin assay (AA) and an antithrombin assay modified by the addition of heparin (H-AA) were performed using sera from healthy subjects, patients predisposed to thrombosis, and patients with thromboembolic disease. Characteristic AA, and H-AA patterns were found in each group. Normal controls and four of 39 "pill" patients (10%) had normal AA and H-AA findings (Pattern A). Normal AA, with "decreasing" H-AA (Pattern B) was found in sera of 31 of 39 "pill" patients (80%) and three patients who had non-embolic arterial occlusive disease. Low AA and "decreasing" H-AA (Pattern C) occurred in sera of four "pill"-takers (10%) and one patient who had an arterial embolus. Low AA and low H-AA (Pattern D) developed in sera of eight patients with thrombophlebitis and seven patients with pulmonary embolus. It is concluded that the AA and H-AA assays help to identify thrombosis-prone (Pattern C) and thrombotic (Pattern D) individuals for whom antiplatelet (aspirin; dipyridamole) or anticoagulant therapy might be beneficial.     

Interactions of plasma proteins with a novel polysaccharide surfactant physisorbed to polyethylene

—A polysaccharide surfactant, dextran-[1,6 bis(2-hydroxypropyl-1-amine)hexane]-dextran, (D-H-D) was prepared by reacting dextran (M_w = 8200) with epichlorohydrin followed by reaction with 1,6-hexanediamine. The D-H-D polymer product was characterized by gel permeation chromatography (GPC), and ¹³C-nuclear magnetic resonance spectroscopy (¹³C-NMR). D-H-D was physisorbed on polyethylene (PE) from aqueous solution, and the adhesion stability and resistance to protein adsorption was examined under static and dynamic flow conditions, using a modified rotating disk system. Modified surfaces were characterized by attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), electron spectroscopy for chemical analysis (ESCA) and by water contact angles. Under applied shear stresses of up to 73 dyn cm^-2, the adhesion of D-H-D on PE was sufficient to inhibit desorption by water (>90% D-H-D on PE was retained) and 5% SDS surfactant solution (approximately 83% D-H-D retained), as determined by ATR-FTIR. Under similar shear stress conditions, albumin adsorption on D-H-D modified PE was reduced by over 90%, and protein adsorption from fresh human plasma was reduced by approximately 70% compared with unmodified PE. The results are discussed in terms of interfacial forces, and the suitability of this approach for studying protein-surface interactions and for developing a novel class of protein-resistant biomaterials.     

The plasma contact system in atopic asthma

An in vitro test for the rate of appearance of kallikrein in plasma due to contact system activation by dextran sulfate at 0 degree C was applied to plasmas of 19 atopic asthma patients and 19 age- and sex-matched controls without atopy. The average prekallikrein activation rate was markedly higher in the plasmas of the atopic patients. Mean endogenous heparin levels were also elevated.     

The procoagulant and proinflammatory plasma contact system

The contact system is a plasma protease cascade that is initiated by coagulation factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via the intrinsic pathway of coagulation or the kallikrein–kinin system, respectively. The biochemistry of the contact system in vitro is well understood, however, its in vivo functions are just beginning to emerge. Data obtained in genetically engineered mice have revealed an essential function of the contact system for thrombus formation. Severe deficiency in contact system proteases impairs thrombus formation but does not reduce the hemostatic capacity of affected individuals. The system is activated by an inorganic polymer, polyphosphate that is released from activated platelets. Excessive inherited activation of the contact system causes a life-threatening swelling disorder, hereditary angioedema. Activation of the contact system by pathogens contributes to leakage in bacterial infections. Mast-cell-derived heparin triggers contact-system-mediated edema formation with implications for allergic disease states. Here we present an overview about the plasma contact system in occlusive and inflammatory disease and its contribution to health and pathology.     

Distinctive regulation of contact activation by antithrombin and C1-inhibitor on activated platelets and material surfaces

Activated human plate lets trigger FXII-mediated contact activation, which leads to the generation of FXIIa–antithrombin (AT) and FXIa–AT complexes. This suggests that contact activation takes place at different sites, on activated platelets and material surfaces, during therapeutic procedures involving biomaterials in contact with blood and is differentially regulated. Here we show that activation in platelet-poor plasma, platelet-rich plasma (PRP), and whole blood induced by glass, kaolin, and polyphosphate elicited high levels of FXIIa-C1-inhibitor (C1INH), low levels of FXIa–C1INH and KK–C1INH, and almost no AT complexes. Platelet activation, in both PRP and blood, led to the formation of FXIIa–AT, FXIa–AT, and kallikrein (KK)–AT but almost no C1INH complexes. In severe trauma patients, FXIIa–AT and FXIa–AT were correlated with the release of thrombospondin-1 (TSP-1) from activated platelets. In contrast, FXIIa–C1INH complexes were detected when the FXIIa–AT levels were low. No correlations were found between FXIIa–C1INH and FXIIa–AT or TSP-1. Inhibition of FXIIa on material surfaces was also shown to affect the function of aggregating platelets. In conclusion, formation of FXIIa–AT and FXIIa–C1INH complexes can help to distinguish between contact activation triggered by biomaterial surfaces and by activated platelets. Platelet aggregation studies also demonstrated that platelet function is influenced by material surface-mediated contact activation and that generation of FXIIa–AT complexes may serve as a new biomarker for thrombotic reactions during therapeutic procedures employing biomaterial devices.     

Blood plasma contact activation on silicon, titanium and aluminium

In the present work, blood plasma protein deposition to spontaneously air oxidized silicon, titanium and aluminium was re-investigated in vitro. Immunological- and null ellipsometry methods were used to detect and quantitate adsorbed proteins, RIA methods to study the retention of preadsorbed 125I-HSA upon exposure to buffer or blood plasma, and kallikrein-specific colorimetric substrate S-2302 to follow the surface generation of kallikrein. The results show that the contact activation of coagulation and complement systems are connected on Si and Ti, but not on Al, via coagulation factor XII. Preadsorbed 125I-HSA was most readily displaced on silicon, followed by titanium and aluminium. The surfaces displayed different antibody binding patterns after short and long-time exposures to plasma. Titanium and silicon bound anti-HMWK after 1 min in plasma, but aluminium did not. When the plasma incubation time was prolonged up to 2h the anti-HMWK binding disappeared totally on titanium and decreased on silicon. During the same time period, anti-C3c binding increased to the three types of surfaces. Also, the anti-C3c binding onto Si and Ti, but not Al, disappeared after incubation in Factor XII deficient plasma or when a specific coagulation factor XII (Factor XII) inhibitor, corn trypsin inhibitor (CTI) was added to normal plasma. The surface contacted plasmas cleaved the kallikrein-specific reagent S-2302 both after single surface contact, and after reincubation of surfaces in fresh plasma. The results show that C3b and Factor XIIa and their degradation products were retained at the surfaces.     

Contributions of contact activation pathways of coagulation factor XII in plasma

Activation of human blood plasma coagulation by contact with hydrophilic or hydrophobic surfaces (procoagulants) is dominated by kallikrein (Kal)-mediated activation of the blood zymogen FXII (Hageman Factor). Mathematical modeling of prekallikrein (PK)-deficient platelet-poor plasma (d(PK)PPP) and PK-reconstituted d(PK)PPP (Rd(PK)PPP) coagulation shows that autoactivation of FXII (FXII-->[surface]FXII) produces no more than about 25% of the total FXIIa produced by the intrinsic pathway. Autoactivation and reciprocal-activation increase in the same proportion with procoagulant surface energy (water-wettability), whereas total amount of FXIIa produced per-unit-area procoagulant remains roughly constant for any particular procoagulant. These results suggest that procoagulant surfaces initiate the intrinsic cascade by producing a bolus of FXIIa in proportion to surface energy or surface area but play no additional role in subsequent molecular events in the cascade. Results further suggest that reciprocal-activation occurs in proportion to the amount of FXIIa produced by the initiating autoactivation step.