Adsorption Of Thrombin Research Articles

Molecular imprinted based microcryogels for thrombin purification

In addition to understanding and explaining the functions of proteins, the need for low-cost, easy and efficient purification methods has been increasing in the field of protein purification, which is also important for enzyme production. In this context, an alternative approach has been developed for the purification of thrombin, which has a crucial role in the hemostatic process, via thrombin imprinted microcryogels that allow reuse and have high selectivity. The characterization studies of the microcryogels were accomplished with micro-computed tomography (µCT), scanning electron microscopy (SEM), optical microscope, surface area measurements (BET analyses) and swelling test measurements. By scanning various parameters affecting thrombin adsorption, the maximum thrombin adsorption capacity (Qmax) was found to be 55.86 mg/g. Also, the selectivity of microcryogels was investigated with the competitive agents and reusability studies were performed. The purity of thrombin was evaluated by Fast Performance Liquid Chromatography (FPLC) method. Experimental results indicated that adsorption of thrombin by the developed microcryogels fit the Langmuir isotherm model (Qmax: 55.86 mg/g, R2: 0.9505) and pseudo-second order for three different thrombin concentrations (R2: 0.9978, R2: 0.9998, R2: 0.9999).

Preparation of magnetic poly(ethylene glycol dimethacrylate-N-Methacryloyl-(L)-glutamic acid) particles for thrombin purification

In this study, magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-glutamic acid) (mPEGDMA-MAGA) particles were prepared by the dispersion polymerization in order to purify thrombin effectively. mPEGDMA-MAGA particles were synthesized by adding different ratios of magnetite (Fe3O4) to the medium in addition to the monomer phases EGDMA and MAGA. The characterization studies of mPEGDMA-MAGA particles were used by fourier transform infrared spectroscopy, zeta size measurement, scanning electron microscopy and electron spin resonance. mPEGDMA-MAGA particles were used in thrombin adsorption studies from aqueous thrombin solutions in both batch and magnetically stabilized fluidized bed (MSFB) system. Maximum adsorption capacity in pH 7.4 phosphate buffer solution is 964 IU/g polymer and 134 IU/g polymer in MSFB system and batch system, respectively. The developed magnetic affinity particles enabled the separation of thrombin from different patient serum samples in one step. It has also been observed that magnetic particles can be used repeatedly without significant reduction in adsorption capacity.

A combined computational and experimental approach predicts thrombin adsorption to zeolites

Robust protein-nanomaterial surface analysis is important, but also a challenge. Thrombin plays an important role in the coagulant activity of protein corona mediated by Ca2+ ion exchanged zeolites. However, the mechanism for this modulation remains unresolved. In this study, we proposed a combined computational and experimental approach to determine the adsorbed sites and orientations of thrombin binding to Ca2+ -exchanged LTA-type (CaA) zeolite. Specifically, fourteen ensembles of simulated annealing molecular dynamics (SAMD) simulations and experimental surface residues microenvironment analysis were used to reduce the starting orientations needed for further molecular dynamics (MD) simulations. The combined MD simulations and procoagulant activity characterization also reveal the consequent corresponding deactivation of thrombin on CaA zeolite. It is mainly caused by two aspects: (1) the secondary structure of thrombin can change after its adsorption on the CaA zeolite. (2) The positively charged area of thrombin mediates the preferential interaction between thrombin and CaA zeolite. Some thrombin substrate sites are thus blocked by zeolite after its adsorption. This study not only provides a promising method for characterizing the protein-nanoparticle interaction, but also gives an insight into the design and application of zeolite with high procoagulant activity.

Aptamer functionalized and reduced graphene oxide hybridized porous polymers SPE coupled with LC-MS for adsorption and detection of human α-thrombin.

In this study, reduced graphene oxide (rGO) hybridized high internal phase emulsions were developed and polymerized as porous carriers for aptamer (5'/5AmMC6/-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3') modification to enrich human α-thrombin from serum. The structure and properties of the materials were confirmed by scanning electron microscope (SEM), Fourier transform infrared spectroscope (FT-IR), and X-ray photoelectron spectra (XPS). The adsorption ability and selectivity were studied and the thrombin was detected with liquid chromatography-mass spectrometry (LC-MS). The adsorption of thrombin onto the sorbent was achieved within 30min and the desorption was realized using 5.0mL of acetonitrile/water (80/20, v/v). The thrombin was quantified by LC-MS according to its characteristic peptide sequence of ELLESYIDGR.

Dual-recognition colorimetric sensing of thrombin based on surface-imprinted aptamer-Fe3O4.

Thrombin plays an essential role in blood coagulation and some physiological and pathological processes. The convenient, rapid, sensitive, and specific detection of thrombin is of great significance in clinical research and diagnosis. Herein, surface molecularly imprinted polymer (MIP) was modified on aptamer-functionalized Fe3O4 nanoparticles (MIP-aptamer-Fe3O4 NP) for thrombin colorimetric assay by taking advantage of the peroxidase-like activity of Fe3O4 NP. With the adsorption of thrombin into imprinted cavities, the exposed surface area of Fe3O4 NP decreased, causing a decrease in its peroxidase-like activity toward 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. On the other hand, the reductive amino acids on the thrombin surface also impeded the oxidation of TMB. Both phenomena caused the light blue color of the sensing solution. Thus, a specifically sensitive colorimetric approach for the visual detection of thrombin was proposed with a linear range and limit of detection of 108.1 pmol L-1-2.7 × 10-5 mol L-1 and 27.8 pmol L-1, respectively. Moreover, due to the double recognition elements of MIP and aptamer, the prepared MIP-aptamer-Fe3O4 NP showed higher selectivity to thrombin than that based on only one recognition element. It is worth noting that no special property (e.g. electrochemical or fluorescence activity) of the template was required in this work. Thus, more template molecules can be easily, selectively, and sensitively detected based on the proposed MIP-aptamer-mimic enzyme colorimetric sensing strategy.

Reversible regulation of thrombin adsorption and desorption based on photoresponsive-aptamer modified gold nanoparticles

In the protein separation, adsorption and desorption of target protein have been using different buffer condition. Different buffer will change the structure and activity of target protein in some cases. This work describes the use of different wavelength light for remote regulation of adsorption and desorption of target protein in the same buffer solutions. A dynamic system that captured and released protein in response to light is reported. Matrix gold nanoparticles and light-responsive affinity ligand comprising thrombin aptamer (APT15), polyethylene glycol linker, and azobenzene-modified complementary sequence were used. UV light induced a trans–cis isomerization of the azobenzene that destabilized the duplex of aptamer and azobenzene-modified complementary sequence, resulting in thrombin binding to aptamer sequence. Visible light irradiation resulted in DNA duplex rehybridization and thrombin released. Our work demonstrates that different light wavelengths effectively regulated the adsorption and desorption of thrombin in the same buffer, and this system also can capture and release prothrombin from plasma with different wavelength light. Furthermore, this method can be widely applied to a variety of different protein separation process.

Protein Sensing with Aptamer Immobilized on an Antifouling Binary Self-Assembled Monolayer

A binary self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) and 3,6-dioxa-8-mercaptooctan-1-ol (DMOL) was formed on a gold electrode as a base for protein aptamer immobilization. The aptamer, specific to hemoglobin, was bound to MUA. A DMOL SAM was used to prevent nonspecific protein adsorption. Electrochemical impedance spectroscopy was performed to determine the electrochemical characteristics of a unary SAM of MUA and a binary SAM consisting of MUA and DMOL. The DMOL SAM prevented adsorption of nonspecific proteins (e.g., thrombin). For the binary SAM, 20:80, 50:50, and 80:20 mol % MUA and DMOL were used. The antifouling effect against nonspecific proteins was enhanced as the ratio of DMOL increased, but the signal intensity (impedance change) decreased. The binary SAM with 50:50 mol % MUA and DMOL showed repelling of nonspecific adsorption of thrombin (70.5% decrease in the impedance change), and it still generated a sufficient signal for target molecules. Additional tests with pure protein samples showed that the presence of DMOL did not affect the aptamer function.

Investigation of Multilayered Protein Adsorption on Carbonate Apatite with a QCM Technique

This study is concerned with multilayered protein adsorption on carbonate apatite (CAp) that is measured with a quartz crystal microbalance (QCM) technique. A carbonate apatite-deposited QCM sensor was prepared by electrophoretic deposition (EPD). On the CAp-deposited QCM sensor, fibrinogen adsorption followed by thrombin adsorption was investigated. The adsorption of fibrinogen on CAp led to a clear decrease in the resonance frequency of the sensor, which means that fibrinogen adsorbed on the CAp-deposited QCM sensor. The adsorbed amount of fibrinogen was larger on CAp than on a pristine sensor (Au). Although successive thrombin adsorption occurred on fibrinogen adlayers on both CAp and Au, the adsorbed amount and its conformation were different depending on the surface.

Affinity electromembrane with covalently coupled heparin for thrombin adsorption

A new composite membrane with electrical conductivity and reactive groups (–COOH) was synthesized by copolymerizing pyrrole-3-carboxylic acid and pyrrole onto nylon membrane via the template method. The ligand, heparin, was covalently coupled onto the composite membrane by activating with N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and modified with ethylenediamine. The as-synthesized affinity electromembrane exhibited a uniform conductivity value of 3.7×10−7S/cm and the content of heparin was approximately 1.897mg/g membrane. By applying an electrical stimulation (−0.5V) to the affinity electromembrane, the adsorption capacity and rate were greatly enhanced. Elution of protein showed desorption ratio was up to 92.7% using 2.0M NaSCN solution as the desorption agent. To investigate the performance of dynamic adsorption of heparin-coupled affinity electromembrane, a membrane cartridge to which electrical stimulation can be applied was designed. The result of dynamic separation showed that the time of breakthrough was delayed from 22min in the absence of electrical stimulation to 31min in the presence of electrical stimulation.

Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin–thrombin complex to minimize the biotinylation of the residues involved in thrombin–boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using 125I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilin’s potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.

Polyurethane modified with an antithrombin‐heparin complex via polyethylene oxide linker/spacers: Influence of PEO molecular weight and PEO‐ATH bond on catalytic and direct anticoagulant functions

A segmented polyurethane (PU) was modified with polyethylene oxides (PEO) of varying molecular weight and end group. The PEO served as linker/spacers to immobilize an antithrombin-heparin (ATH) anticoagulant complex on the PU. Isocyanate groups were introduced into the PU to enable attachment of either "conventional" homo-bifunctional dihydroxy-PEO (PEO-OH surface) or a hetero-bifunctional amino-carboxy-PEO (PEO-COOH surface). The PEO surfaces were functionalized with N-hydroxysuccinimide (NHS) groups using appropriate chemistries, and ATH was attached to the distal NHS end of the PEO (PEO-OH-ATH and PEO-COOH-ATH surfaces). Water contact angle and fibrinogen adsorption measurements showed increased hydrophilicity and reduced fibrinogen adsorption from buffer on all PEO surfaces compared to unmodified PU. ATH uptake on NHS-functionalized PEO was quantified by radiolabeling. Despite the different PEO molecular weights and end groups, and NHS-functionalization chemistries, the surface densities of ATH were similar. The adsorption of fibrinogen and antithrombin (AT) from plasma was measured in a single experiment using dual radiolabeling. On PEO-ATH surfaces fibrinogen adsorption was minimal while AT adsorption was high showing the selectivity of the heparin moiety of ATH for AT. The PEO-COOH-ATH surfaces showed slightly greater AT adsorption than the PEO-OH-ATH surfaces. Thrombin adsorption on all of the PEO-ATH surfaces was greater than on the corresponding PEO surfaces without ATH, and was highest on the PEO-OH-ATH, suggesting potential anticoagulant properties for this surface via direct thrombin inhibition by the AT portion of ATH.

On-Chip Synthesis of RNA Aptamer Microarrays for Multiplexed Protein Biosensing with SPR Imaging Measurements

Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.

Near Infrared Surface Plasmon Resonance Phase Imaging and Nanoparticle-Enhanced Surface Plasmon Resonance Phase Imaging for Ultrasensitive Protein and DNA Biosensing with Oligonucleotide and Aptamer Microarrays

The techniques of surface plasmon resonance-phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have been implemented for the multiplexed bioaffinity detection of proteins and nucleic acids. The SPR-PI experiments utilized a near-infrared 860 nm light emitting diode (LED) light source and a wedge depolarizer to create a phase grating on a four-element single-stranded DNA (ssDNA) microarray; bioaffinity adsorption onto the various microarray elements was detected via multiplexed real time phase shift measurements. In a first set of demonstration experiments, an ssDNA aptamer microarray was used to directly detect thrombin at concentrations down to 100 pM with SPR-PI. Two different ssDNA aptamers were used in these experiments with two different Langmuir adsorption coefficients, K(A1) = 4.4 × 10(8) M(-1) and K(A2) = 1.2 × 10(8) M(-1). At concentrations below 1 nM, the equilibrium phase shifts observed upon thrombin adsorption vary linearly with concentration with a slope that is proportional to the appropriate Langmuir adsorption coefficient. The observed detection limit of 100 pM is approximately 20 times more sensitive than that observed previously with SPRI. In a second set of experiments, two short ssDNA oligonucleotides (38mers) were simultaneously detected at concentrations down to 25 fM using a three-sequence hybridization format that employed 120 nm DNA-modified silica nanoparticles to enhance the SPR-PI signal. In this first demonstration of nanoparticle-enhanced SPR-PI, the adsorbed silica nanoparticles provided a greatly enhanced phase shift upon bioaffinity adsorption due to a large increase in the real component of the interfacial refractive index from the adsorbed nanoparticle. As in the case of SPR-PI, the detection limit of 25 fM for nanoparticle-enhanced SPR-PI is approximately 20 times more sensitive than that observed previously with nanoparticle-enhanced SPRI.

Application of MS-Based Proteomics to Study Serum Protein Adsorption/Absorption and Complement C3 Activation on Poly(ethylene glycol) Hydrogels

Although the interaction between cells and poly(ethylene glycol) (PEG) hydrogels is well documented, there lacks a thorough investigation into the adsorption of blood proteins on these surfaces which dictates the observed cellular and in vivo host response. Thus, a clear understanding of how surface-bound proteins mediate the unique biological property of PEG hydrogels is fundamentally important. The information obtained will also provide insights into future biomaterial design. In this study, several mass-spectrometrybased proteomic tools coupled with complementary immunoassays were employed to survey the complex surface-bound serum proteome. The adsorption of vitronectin, thrombin, fibrinogen and complement component C3 was significantly lower on PEG hydrogels than on tissue culture polystyrene (TCPS). Although PEG hydrogels mediated lower C3 adsorption than TCPS, the extent of C3 activation between the two surfaces was comparable. Adherent monocyte density was also significantly lower on PEG hydrogels as compared to TCPS. Taken together, these results support the critical role of the complement C3 in mediating monocyte adhesion on biomaterials. Thus we conclude that the biocompatibility of PEG hydrogels both in vitro and in vivo can be partly contributed to their limited C3 interaction and monocyte activity.

The effect of immobilization of thrombin inhibitors onto self-assembled monolayers on the adsorption and activity of thrombin

Thrombus formation is the major problem associated with biomaterials for blood contact medical devices. The immobilization of inhibitors to thrombin, a serine protease that plays a central role on the coagulation system, on the surface of biomaterials should be a good strategy to avoid blood clotting and increase their hemocompatibility. The aim of this work is the design of a nanostructured surface with capacity to adsorb and inactivate thrombin. The pentapeptide sequence d-Phenylalanine–Proline–Arginine–Proline–Glycine (fPRPG), that was described as a thrombin inhibitor, was immobilized onto tetra(ethylene glycol) terminated self-assembled monolayers (EG4-SAMs). Surface containing different amounts of fPRPG were prepared using different concentrations of N,N′-Carbonyldiimidazole (CDI) during immobilization. The efficiency of fPRPG immobilization was followed using ellipsometry, contact angle measurements, Infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS). Thrombin adsorption was quantified using radiolabelled thrombin and its activity in solution and after adsorption on the developed surfaces was assessed using a chromogenic assay. It was found that, although the immobilization of fPRPG on to EG4-SAMs does not increase its selectivity to thrombin, the activity of the adsorbed thrombin was inhibited in a peptide concentration dependent way. We concluded that SAMs with fPRPG immobilized in high amounts can be used as thrombin-inhibitor surfaces, which is a good step on the development of new surfaces for blood contact devices.

Synthesis and Characterization of Thrombin Conjugated γ‐Fe2O3 Magnetic Nanoparticles for Hemostasis

AbstractThrombin is the final protease produced in the clotting pathways. Thrombin has been used in the clinic more than six decades for topical hemostasis and wound management. In human plasma the half‐life of thrombin is shorter than 15 seconds due to close control by inhibitors. In order to stabilize thrombin, this enzyme was conjugated covalently and physically to γ‐Fe2O3 magnetic nanoparticles. The physical conjugation was accomplished through adsorption of thrombin to BSA coating on the nanoparticles. The coagulant activity of the covalently bound thrombin was significantly lower than that of the physically adsorbed thrombin. Leakage of the physically bound thrombin into PBS containing 4% HSA was negligible. The physical conjugation of thrombin onto the nanoparticles stabilized the thrombin against its major inhibitor antithrombin III and improved its storage stability. At optimal CaCl2 concentration, the clotting time by the bound thrombin is shorter than that of the free enzyme. This novel conjugated thrombin may be an efficient candidate for topical hemostasis and wound healing.

Bivalent Binding to γA/γ′-Fibrin Engages Both Exosites of Thrombin and Protects It from Inhibition by the Antithrombin-Heparin Complex

Thrombin exosite 1 binds the predominant gamma(A)/gamma(A)-fibrin form with low affinity. A subpopulation of fibrin molecules, gamma(A)/gamma'-fibrin, has an extended COOH terminus gamma'-chain that binds exosite 2 of thrombin. Bivalent binding to gamma(A)/gamma'-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from gamma(A)/gamma'-fibrin clots than from gamma(A)/gamma(A)-fibrin clots. After 24 h of washing, however, both gamma(A)/gamma'- and gamma(A)/gamma(A)-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in gamma(A)/gamma'-fibrin clots than they were in gamma(A)/gamma(A)-fibrin clots. This difference reflects bivalent binding of thrombin to gamma(A)/gamma'-fibrin because (a) it is abolished by addition of a gamma'-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the gamma'-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with gamma(A)/gamma'-fibrin than with gamma(A)/gamma(A)-fibrin clots. Thus, bivalent binding of thrombin to gamma(A)/gamma'-fibrin protects thrombin from inhibition, raising the possibility that gamma(A)/gamma'-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.

Novel thromboresistant materials

The development of a clinically durable small-diameter vascular graft as well as permanently implantable biosensors and artificial organ systems that interface with blood, including the artificial heart, kidney, liver, and lung, remain limited by surface-induced thrombotic responses. Recent breakthroughs in materials science, along with a growing understanding of the molecular events that underlay thrombosis, has led to the design and clinical evaluation of a variety of biologically active coatings that inhibit components of the coagulation pathway and platelet responses by surface immobilization or controlled release of bioactive agents. This report reviews recent progress in generating synthetic thromboresistant surfaces that inhibit (1) protein and cell adsorption, (2) thrombin and fibrin formation, and (3) platelet activation and aggregation.

Inhibition of Thrombin Cleavage of Factor VIII by Fibrinogen γ' Chain Carboxyl Terminus.

Fibrinogen can contain two types of γ chains, γA and γ’, that have identical sequences for the first 407 amino acids, but differ at their carboxyl termini. The γA chain contains four amino acids from γA 408–411, whereas the γ’ chain contains a different, highly anionic sequence of twenty amino acids from γ’ 408–427. This small change results in profound differences in the fibrinogen isoforms containing these distinct chains. The γ’ chain contains high affinity binding sites for zymogen factor XIII and for thrombin, and fibrinogen containing γ’ chains forms fibrin clots that are resistant to fibrinolysis. We have shown previously that the fibrinogen γ’ chain carboxyl terminus binds to thrombin exosite II, an anion-binding exosite known for its heparin binding activity. Recent reports show that the presence of the γ’ chain in fibrinogen reduces prothrombin activation in whole plasma assays. This inhibition may correspond to an activity originally described by Seegers in 1945 as “antithrombin I”, the adsorption of thrombin to fibrin. We have therefore investigated the enzymatic effects of γ’ peptide binding to thrombin in order to determine the role of the γ’ chain in the inhibition of prothrombin activation. In whole plasma, the γ’410–427 peptide prolonged the activated partial thromboplastin time (aPTT) in a dose-dependent manner, causing a delay in clotting from 29 seconds to 47 seconds at a concentration of 500 μM. A scrambled control peptide at the same concentration had little effect, increasing the clotting time to only 31.4 seconds. A series of γ’ deletion peptides were tested for their ability to prolong the aPTT. The inhibitory effect of the peptides corresponded in rank order to their thrombin binding affinity. In contrast to the aPTT, the prothrombin time increased only slightly from 11.7 seconds to 12.8 seconds (INR from 1.04 to 1.14) with the γ’410–427 peptide. This suggested that the γ’ peptide exerted its inhibitory effect on (a) component(s) of the intrinsic pathway, rather than the extrinsic pathway. We therefore investigated the effect of the γ’ peptide on thrombin activation of factor VIII, a thrombin substrate that, unlike factor V, is unique to the intrinsic pathway. Using purified factor VIII and thrombin in vitro, we showed that the γ’ peptide inhibited thrombin cleavage of factor VIII. However, this inhibitory effect was not due to direct inhibition of the thrombin active site, since the free γ’ peptide had little effect on thrombin cleavage of a small peptidyl substrate, tosyl-glycyl-prolyl-arginine-4-nitranilide acetate. These results suggest that factor VIII interactions with thrombin exosite II are essential for efficient cleavage of factor VIII, and support recent findings by others that interactions between thrombin exosite II and the A2 domain of factor VIII facilitate thrombin-catalyzed cleavage. These findings may explain, at least in part, the inhibitory effect of the γ’ chain on prothrombin activation in plasma.

Thrombin, kallikrein and complement C5b-9 adsorption on hydrophilic and hydrophobic titanium and glass after short time exposure to whole blood

Hydrophilic and hydrophobic titanium and glass were exposed to capillary whole blood between 5 s and 24 h. The time-sequence for adsorption of thrombin, kallikrein and complement C5b-9, and their relationship with adherent platelets and polymorphonuclear granulocyte (PMN) activation were investigated. Adsorbed thrombin and kallikrein were measured by cleavage of specific chromogenic substances, S-2238 and S-2303, respectively. Complement C5b-9 and expression of CD11b, CD66b, CD62P and Pan-platelets were measured by immunofluorescence. Thrombin and kallikrein were present on the surfaces during the whole investigated periods. Platelet adhesion and PMN cell adhesion and activation on all surfaces and activation of platelets on hydrophobic surfaces showed a similar pattern to thrombin adsorption. Kallikrein adsorption had a different pattern on each surface. C5b-9 was detected between 32 min and 24 h of blood exposure and a varying pattern of C5b-9 coverage was observed on each surface. In conclusion, our results indicate that the interaction between material and blood coagulation and kinin-activating proteins regulate the adhesion and activation of blood cells, whereas after longer time the coagulation and kallikrein-kinin system play minor roles and the complement system is decisive for mediating and elongating the inflammatory process.