The effect of adsorbed fibrinogen, fibronectin, von Willebrand factor and vitronectin on the procoagulant state of adherent platelets

Procoagulant (activated) platelets provide a site for assembly of the prothrombinase complex which can rapidly convert prothrombin into thrombin (a potent inducer of clot formation). Previously, we reported that adhesion of platelets to surfaces preadsorbed with blood plasma caused them to become procoagulant. In the present study we investigated the effect of adsorbed adhesion proteins (fibrinogen (Fg), fibronectin (Fn), von Willebrand factor (vWF) and vitronectin (Vn)) on the procoagulant activity of adherent platelets. Adsorbed Fn, vWF and Fg promoted platelet adhesion in the following order: Fn < vWF = Fg. However, these proteins promoted platelet activation (thrombin generation per adherent platelet) in the following order: Fg < Fn < vWF. Adsorption with a series of dilutions of normal plasma, serum, and plasmas deficient in or depleted of von Willebrand factor (de-vWF), fibronectin (de-Fn), vitronectin (de-Vn), or both vitronectin and fibronectin (de-VnFn) resulted in varied platelet adhesion, but little difference in platelet activation. However, preadsorption with dilute de-vWF plasma induced lower procoagulant activity than normal plasma. Preadsorption with normal plasma resulted in higher levels of platelet activation than preadsorption with Fg, suggesting that adsorption of plasma proteins other than Fg caused the high levels of activation observed for plasma preadsorbed surfaces.     

Glow discharge plasma treatment of polyethylene tubing with tetraglyme results in ultralow fibrinogen adsorption and greatly reduced platelet adhesion

Previous studies from our lab have shown that fibrinogen adsorption (Gamma(Fg)) must be reduced below 10 ng/cm(2) to significantly reduce platelet adhesion, and that radio frequency glow discharge (RFGD) treatment of polymeric films in the presence of tetraethylene glycol dimethyl ether (tetraglyme) can reduce Gamma(Fg) to the desired ultralow value. In this report, the effects of RFGD coatings of tetraglyme on the lumenal surface of PE tubing on Gamma(Fg) and on blood interactions both in vitro and ex vivo are described. Gamma(Fg) on the tetraglyme-coated PE tubing was reduced to the desired ultralow level (<10 ng/cm(2)), and we also observed a significant decrease in adsorption of von Willebrand's factor. In vitro platelet adhesion from washed platelet suspensions, platelet rich plasma, or whole blood to tetraglyme-coated PE tubing was decreased compared to PE, polyurethane, or silicone rubber tubes. In addition, thrombin generation by platelets adherent to tetraglyme-coated PE was also much less than by platelets adherent to PE. When inserted in an ex vivo carotid artery-carotid artery shunt in sheep, the RFGD tetraglyme-coated PE exhibited a very low number of adherent platelets compared to heparin-coated, chromic acid-etched, or plain PE. The RFGD tetraglyme-coated PE tubes exhibited high protein and platelet resistance in vitro, and high platelet resistance ex vivo. The improved hemocompatibility is attributed to the unique chemical structure of RFGD tetraglyme that makes it highly protein resistant.     

Platelet adhesion to radiofrequency glow-discharge-deposited fluorocarbon polymers preadsorbed with selectively depleted plasmas show the primary role of fibrinogen

Fluorocarbon radio-frequency glow-discharge (RFGD) treatment has previously been shown to cause decreased platelet adhesion despite the presence of adsorbed fibrinogen on the surfaces. In this study platelet adhesion to fluorocarbon RFGD-treated surfaces preadsorbed with human plasma was further examined. A series of plasma deposited fluorocarbon thin films were made by varying the C3F6/CH4 ratio in the monomer feed. The surfaces were preadsorbed with plasma, serum, or plasma selectively depleted of fibronectin, vitronectin, or Von Willebrand factor, and platelet adhesion was measured. We also measured fibrinogen adsorption to the surfaces from plasma, monoclonal antibody binding to adsorbed fibrinogen and SDS elutability of the adsorbed fibrinogen. The antibodies used bind to the three putative platelet binding sites on fibrinogen, namely, M1 antibody binds to the dodecapeptide at the C-terminus of the gamma chain, gamma (402-411), R1 antibody binds to a sequence in the Aalpha chain (87-100) which includes RGDF at Aalpha (95-98) and R2 antibody binds a sequence in the Aalpha chain (566-580) which includes RGDS at Aalpha (572-575). Fibrinogen was found to play a decisive role in mediating platelet adhesion to the fluorocarbon surfaces contacting plasma. Few platelets adhered to the fluorocarbon surfaces preadsorbed with serum, while preadsorption with plasma selectively-depleted of either fibronectin, vitronectin, or von Willebrand factor did not decrease platelet adhesion significantly. Replenishment of exogenous fibrinogen to serum restored platelet adhesion, while replenishment of the other proteins had no effect. Platelet adhesion to the fluorocarbon surfaces was lower than to PET or the methane glow-discharge-treated PET. However, there was no apparent correlation between platelet adhesion and the amount of fibrinogen adsorption or monoclonal antibody binding to surface-bound fibrinogen.     

Adhesion and aggregation of thrombin prestimulated human platelets: evaluation of a series of biomaterials characterized by ESCA

Five materials of interest in blood contact applications (PVC, Silastic, Biomer and siliconized glass) were internally coated on glass tubes and exposed to suspensions of platelets and red cells. Uncoated glass was also examined. Thrombin was added (prestimulation) to some suspensions before exposure to the biomaterial surfaces. The three polymeric surfaces were characterized by electron spectroscopy for chemical analysis (ESCA). Prestimulation with thrombin leads to increased adhesion of single platelets only with Silastic. With a wide range of surface types, thrombin prestimulation consistently leads to higher levels of platelet accumulation in the form of aggregates; the PVC-coated material showed the highest levels. ESCA analysis of PVC, however, suggested our coating was impure or an oxidized material.     

Ex vivo and in vitro platelet adhesion on RFGD deposited polymers.

Clinical applications of small-diameter synthetic vascular grafts are hindered by their highly thrombogenic surfaces. To develop vascular grafts that resist thrombotic occlusion, a radio frequency glow discharge (RFGD) process was employed to modify the surface of existing graft materials. Ultrathin coatings of RFGD polymers of ethylene (E), tetrafluoroethylene (TFE), and hexamethyldisiloxane (HMDS) were deposited on the lumen of Dacron grafts. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA). The effect of glow discharge treatments on platelet-graft interactions was evaluated in an ex vivo baboon shunt model. Following placement of an untreated or RFGD-treated graft in the shunt, deposition of 111Indium-labeled platelets was monitored for 60 min by gamma camera imaging. Untreated Dacron rapidly accumulated large numbers of platelets, reaching a plateau in 60 min. HMDS- and TFE-treated Dacron had significantly lower levels of platelet deposition compared to the untreated control. In contrast, the ethylene treatment of Dacron augmented platelet deposition, making it the most platelet-adherent surface studied. In vitro studies were also performed using untreated and RFGD-treated poly (ethylene terephthalate) (PET) coverslips. ESCA verified that the surface composition of the untreated and RFGD-treated coverslips were virtually identical to their untreated and treated Dacron graft counterparts. Samples were incubated in washed baboon platelet suspensions for 2 h at 37 degrees C. Platelet adhesion on the untreated PET was relatively high, and many of the platelets had a completely spread morphology. The HMDS and TFE treatment of PET reduced the number of adherent platelets and prevented platelet spreading on the surface. Platelet adhesion and spreading on the ethylene-treated surface was the highest among the four studied. There is a remarkable linear correlation of the ex vivo and in vitro platelet adhesion data.     

Thrombin generation in platelet-rich plasma as a tool for the detection of hypercoagulability in young stroke patients

The time course of the concentration of active thrombin in clotting plasma (the thrombogram) was measured by subsampling from platelet-rich plasma (PRP) and continuous chromogenic measurement of platelet-poor plasma (PPP) in 41 stroke patients under the age of 50, in whom stroke could not be attributed to cardioembolic disease, arterial dissection or vasculitis. A significant increase in the area under the thrombogram (endogenous thrombin potential, ETP) was seen in 23 patients. In 9 of them, ETP was increased in PRP but normal in PPP. High ETP in PRP was significantly associated with stroke, both in the middle and in the highest tercile of the ETP (odds ratio 5.1, range 1.8-15.1, and 3.7, range 1.3-10.3, respectively). A decreased sensitivity to the inhibitory action of thrombomodulin (TM) on thrombin generation was observed in 5 of 37 cases. No further definition of the cause of increased thrombin generation or TM resistance was attempted, except for the role of von Willebrand factor (vWF). ETP in PRP, platelet-derived procoagulant activity and vWF were correlated and higher in patients than in controls (p=0.002, p=0.045 and p=0.0006, respectively). This confirms the correlation between vWF level and stroke at young age found in epidemiological studies. It suggests that the role of vWF in thrombin generation, which has been demonstrated in vitro, may be the underlying mechanism of this correlation. In summary, hypercoagulability, defined as an increased capacity of the platelet plasma system to form thrombin, is found in over half of the patients under 50 years with an otherwise unexplained stroke. Sometimes it is due to increased plasma factor activity, sometimes to an increased procoagulant activity of the platelets.     

Co-adsorbed fibrinogen and von Willebrand factor augment platelet procoagulant activity and spreading

Previously we observed that platelets adherent to surfaces preadsorbed with blood plasma exhibited 1.3 to 2.4 times greater procoagulant activity than platelets on surfaces adsorbed with fibrinogen (Fg) only. These observations suggested that the adhesion proteins adsorbed from plasma may activate platelets in a cooperative, or synergistic manner. In the present study, polystyrene surfaces adsorbed with both Fg and vWF induced up to three times greater procoagulant activity than surfaces adsorbed with Fg or vWF only. The amounts of Fg and vWF adsorbed from binary mixtures that resulted in increased procoagulant activity were found to be similar to the amounts that adsorbed to PS from 100% plasma. The effect of adsorbed adhesion proteins on platelet spreading was also investigated. The proportion of fully spread platelets increased, depending on the adhesion protein preadsorbed to the surface, in the following order: vWF < Fg < Fn < (vWF + Fg) < Vn < plasma.     

Facilitating roles of murine platelet glycoprotein Ib and αIIbβ3 in phosphatidylserine exposure during vWF–collagen-induced thrombus formation

Vessel wall damage exposes collagen fibres, to which platelets adhere directly via the collagen receptors glycoprotein (GP) VI and integrin α₂β₁ and indirectly by collagen-bound von Willebrand factor (vWF) via the GPIb-V-IX and integrin αIIbβ3 receptor complexes. Platelet–collagen interaction under shear stimulates thrombus formation in two ways, by integrin-dependent formation of platelet aggregates and by surface exposure of procoagulant phosphatidylserine (PS). GPVI is involved in both processes, complemented by α2β1. In mouse blood flowing over collagen, we investigated the additional role of platelet–vWF binding via GPIb and αIIbβ3. Inhibition of GPIb as well as blocking of vWF binding to collagen reduced stable platelet adhesion at high shear rate. This was accompanied by delayed platelet Ca²⁺ responses and reduced PS exposure, while microaggregates were still formed. Inhibition of integrin αIIbβ3 with JON/A antibody, which blocks αIIbβ3 binding to both vWF and fibrinogen, reduced PS exposure and aggregate formation. The JON/A effects were not enhanced by combined blocking of GPIb–vWF binding, suggesting a function for αIIbβ3 downstream of GPIb. Typically, with blood from FcR γ-chain +/− mutant mice, expressing 50% of normal platelet GPVI levels, GPIb blockage almost completely abolished platelet adhesion and PS exposure. Together, these data indicate that, under physiological conditions of flow, both adhesive receptors GPIb and αIIbβ3 facilitate GPVI-mediated PS exposure by stabilizing platelet binding to collagen. Hence, these glycoproteins have an assistant procoagulant role in collagen-dependent thrombus formation, which is most prominent at reduced GPVI activity and is independent of the presence of thrombin.     

Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition

The ability of tetraethylene glycol dimethyl ether (tetraglyme) plasma deposited coatings exhibiting ultralow fibrinogen adsorption to reduce blood activation was studied with six in vitro methods, namely fibrinogen and von Willebrand's factor adsorption, total protein adsorption, clotting time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed flow catheter model. Surface plasmon resonance results showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the intrinsic coagulation cascade. Platelet adhesion and thrombin generation by adherent platelets were greatly reduced on tetraglyme-coated materials, compared with uncoated and Biospan-coated glass slides. In the in vitro disturbed blood flow model, tetraglyme plasma coated catheters had 50% less thrombus than did the uncoated catheters. Tetraglyme-coated materials thus had greatly reduced blood interactions as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only due to their reduced platelet adhesion and activation, but also to a generalized reduction in blood interactions.     

Effects of VLDL and remnant particles on platelets

There is a considerable body of evidence supporting an association between hypertriglyceridaemia, a hypercoagulable state and atherothrombosis. A disorder of triglyceride metabolism is a key feature of the metabolic syndrome that increases risk of both ischaemic heart disease and type 2 diabetes approximately 3-fold. An increasing prevalence of obesity and metabolic syndrome is likely to contribute markedly to the prevalent ischaemic heart in the foreseeable future, and therefore it is crucial to understand mechanisms linking hypertriglyceridaemia and a hypercoagulable state. Activation of platelets and the coagulation cascade are intertwined. VLDL and remnant lipoprotein concentrations are often increased with the metabolic syndrome. These lipoproteins have the capacity to activate platelets and the coagulation pathway, and to support the assembly of the prothrombinase complex. VLDL also upregulates expression of the plasminogen activator inhibitor-1 gene and plasminogen activator inhibitor-1 antigen and activity, a process accompanied by platelet aggregation and clot formation. The surface membrane of activated platelets also supports the assembly and activity of the prothrombinase complex, resulting in further thrombin generation and amplification of the coagulation cascade. Fibrinolysis is also less efficient when thrombin is generated. Thrombin induces thrombin activatable fibrinolysis inhibitor. Thrombin activatable fibrinolysis inhibitor is a carboxypeptidase that cleaves the carboxylic lysine residues on fibrin, thereby abolishing the critical binding site for tPA-plasminogen decreasing plasmin formation. Thus the evidence is supportive of dysregulated coagulation, and impaired fibrinolysis with a predisposition to atherothrombosis, in conditions such as the metabolic syndrome, in which there are increased concentrations of VLDL and remnant lipoproteins. The purpose of this review is to describe the current evidence supporting a procoagulant state induced by VLDL and remnant lipoproteins. The role of these lipoprotein classes in (1) platelet activation; (2) the intrinsic coagulation cascade, and (3) clot formation and fibrinolysis is discussed.     

含氟聚醚聚氨酯与聚醚聚氨酯共混的表面结构及血液相容性的研究

为了获得一种高氟聚氨酯表面 ,进一步改善聚醚聚氨酯的生物相容性和生物稳定性 ,将侧链含氟聚氨酯与聚醚聚氨酯共混而实现这一目的。通过 XPS、AFM、接触角和血小板黏附对含氟聚醚聚氨酯和聚醚聚氨酯共混物表面结构和血液相容性进行研究发现 ,在聚醚聚氨酯共混入极少量的氟 (0 .342 wt% )就能具有与含氟聚醚聚氨酯相同的表面结构和良好的血液相容性 ,而且共混物表面的强疏水性和对血小板的黏附与体系中混入的含氟聚醚聚氨酯的量无关 ,与表面 CF3的含量有关     

Effects of fibrinogen residence time and shear rate on the morphology and procoagulant activity of human platelets adherent to polymeric biomaterials

Fibrinogen readily adsorbs to the surface of biomaterials and, because of its demonstrated ability to support platelet adhesion and aggregation, plays a role in thrombotic events associated with the implantation of synthetic materials in the human body. Thus, understanding the factors influencing the interactions of fibrinogen with biomaterials, and how platelet responses are affected, is crucial for the development of synthetic materials exhibiting improved blood compatibility. In this study, the effects of fibrinogen residence time and shear rate on the procoagulant activity of adherent platelets, along with their morphologic status, as deduced from scanning electron microscopy, were investigated. To examine whether adherent platelets promoted the generation of thrombin, polymeric materials (polytetrafluoroethylene, polyethylene, and silicone rubber) preadsorbed with fibrinogen were exposed to platelet suspensions at different wall shear rates and then incubated with clotting factors for 5 minutes under static conditions. The amount of thrombin generated per platelet was calculated from the optical density of the color developed by adding substrate S-2238. Scanning electron microscopy images of the platelets revealed that the platelets exhibited different morphologies, depending on the shear rate and residence time of the adsorbed fibrinogen. Platelets ranged from their normal discoid shape observed primarily under static conditions, to that of fully spread platelets. Results from this study show that platelets, in the presence of shear forces, undergo activation on exposure to surfaces on which adsorbed fibrinogen has resided for short residence times rather than long residence times. Interestingly, studies examining the procoagulant responses of such adherent platelets demonstrated that the platelets attached to the fibrinogen coated materials did not promote significant thrombin generation. Such low prothrombinase activity of adherent platelets suggests that adsorbed fibrinogen, while capable of supporting platelet adhesion and spreading on biomaterials, does not necessarily enhance the procoagulant activity of adherent platelets.     

The role of phospholipids in platelet function

Platelet phospholipids undergo significant alterations during aggregation induced by thrombin or other agents. There is an early increase in phosphatidic acid, with a decrease in phosphatidyl inositol. De novo synthesis of most phospholipids from 14C-glycerol is decreased. Thrombin stimulates 32P-phosphate incorporation into di- and triphosphoinositides, suggesting increased phosphorylation of phosphatidyl inositol during aggregation. Arachidonic acid for prostaglandin synthesis is released from platelet phospholipids. Thrombin induced aggregation results in release of arachidonic acid primarily from phosphatidyl choline and phosphatidyl inositol. The availability of free arachidonic acid may be regulated by platelet phospholipase A2 activity. The latter activity is stimulated by thrombin, requires calcium ions, and is inhibited by agents which elevate cyclic adenosine monophosphate. Phospholipids are probably an essential component of the platelet surface lipoprotein procoagulant activity known as platelet factor 3. There is evidence that calcium ions may mediate binding between gamma carboxyglutamic acid residues on the amino terminal portion of prothrombin and negatively charged phosphate groups on phospholipid micelles. Binding of prothrombin to phospholipid on the platelet surface may orient the former such as to facilitate the prothrombinase activity of Factor Xa. Platelet phospholipids and platelet factor 3 activity are decreased in some congenital and myeloproliferative disorders. Increases in these factors may be associated with thrombotic and arterial occlusive disorders.     

Biocompatibility of fluorinated poly(organophosphazene)

We investigated neutrophil and platelet adhesion on a fluorinated poly(organophosphazene) in vitro. The results suggested that neutrophil and platelet adhesion on the poly(organophosphazene) only occurred on a few occasions, as observed by SEM. We demonstrated that the fluorinated poly(organophosphazene) showed excellent biocompatibility compared with the poly(organophosphazene) without the fluorinated side groups or PDMS. Additionally, we estimated the competitive plasma protein adsorption to the fluorinated poly(organophosphazene) using a gold-colloid-labeled immunoassay. Interestingly, the fluorinated poly(organophosphazene) film selectively adsorbed albumin when compared with gamma-globulin and fibrinogen, suggesting that a selective albumin adsorption on the film is responsible for the suppression of platelet adhesion.     

Biocompatibility of fluorinated poly(organophosphazene)

We investigated neutrophil and platelet adhesion on a fluorinated poly(organophosphazene) in vitro. The results suggested that neutrophil and platelet adhesion on the poly(organophosphazene) only occurred on a few occasions, as observed by SEM. We demonstrated that the fluorinated poly(organophosphazene) showed excellent biocompatibility compared with the poly(organophosphazene) without the fluorinated side groups or PDMS. Additionally, we estimated the competitive plasma protein adsorption to the fluorinated poly(organophosphazene) using a gold-colloid-labeled immunoassay. Interestingly, the fluorinated poly(organophosphazene) film selectively adsorbed albumin when compared with γ-globulin and fibrinogen, suggesting that a selective albumin adsorption on the film is responsible for the suppression of platelet adhesion.     

Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading. An immunoelectron microscopy study.

The authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton, and 3) during the early stage of platelet activation, a communication exists between the alpha granules and the platelet surface.     

Thrombogenicity of polysaccharide-coated surfaces

Heparinization of artificial surfaces has been proven to reduce the intrinsic thrombogenicity of such surfaces. The mechanism by which immobilized heparin reduces thrombogenicity is not completely understood. In the present study heparin-, alginic acid- and chondroitin-6-sulphate-coated surfaces were examined for protein adsorption, platelet adhesion and thrombin generation. The protein-binding capacity from solutions of purified proteins was significantly higher for heparin-coated surfaces when compared with alginic acid- and chondroitin sulphate-coated surfaces. Yet, when the surfaces were exposed to flowing plasma, only the heparinized surface adsorbed significant amounts of antithrombin. None of the surfaces adsorbed fibrinogen under these conditions, and as a result no platelets adhered from flowing whole blood. Our results indicate that protein adsorption and platelet adhesion from anticoagulated blood cannot be used to assess the thrombogenicity of (coated) artificial surfaces. Indeed, the thrombin generation potentials of the different surfaces varied remarkable: while non-coated surface readily produced thrombin, alginic acid- and chondroitin sulphate-coated surfaces showed a marked reduction and virtually no thrombin was generated in flowing whole blood passing by heparinized surfaces.     

Influence of factor V HR2 on thrombin generation and clinical manifestation in rare bleeding disorders

In this study we investigated the influence of the presence of the factor V HR2 haplotype, defined by the factor V gene mutation H1299R (FV(HR2)), on thrombin generation. Measurements were performed in platelet-poor plasma of individuals with factor V(HR2) or factor V(Leiden) in comparison to a control group carrying none of these mutations. Coagulation was triggered by low concentrations of recombinant tissue factor in the presence of activated protein C. Thrombin generation was monitored by a fluorogenic substrate. The endogenous thrombin potential was calculated from the obtained curves. As a result we observed an increased thrombin generation both for individuals heterozygous and homozygous for FV(HR2). The level of endogenous thrombin potential is in the same range as in samples of patients heterozygous or homozygous for FV(Leiden). The results indicate that FV(HR2) plays a role as a risk factor for venous thrombosis in homozygous patients through an increased thrombin generation. The association between different clinical manifestations in individuals with FVII deficiency and endogenous thrombin potential and the presence of FV(HR2) was studied.     

Platelet adhesion and cellular interaction with poly(ethylene oxide) immobilized onto silicone rubber membrane surfaces

Cellular interaction and platelet adsorption were investigated on poly(ethylene oxide) (PEO) immobilized silicone rubber membrane (SR) which has polyacrylic acid grafts on the surfaces. Polyacrylic acid (PAA) had been introduced to the SR surface after Ar plasma treatment of SR surfaces to introduce peroxide groups. Surface characterizations were made using ATR-FTIR, ESCA, SEM, and contact angle measurements. Experimental results obtained by ESCA high resolution curve fitting spectra indicated that the amount of bisamino PEO of different molecular weights immobilized onto SR surfaces were similar, which showed that the influence of the length of molecular chains (-C-C-O-) on the reactivity of terminal amino group is negligible. The wettability of modified SR surfaces increased with an increase in PEO molecular weight. Biological studies such as corneal epithelial cell culture and blood platelet adhesion were performed to understand the biocompatibility of modified SR surfaces. Biological studies using corneal epithelial cells showed that cell migration, attachment and proliferation onto PEO-20000 immobilized SR surface were suppressed, whereas these biological activities on PEO-600 were enhanced. Another study on platelet adhesion revealed that many platelets attached to PEO-600 immobilized SR, while platelet deposition was rarely observed on SR grafted with PEO-3350. The effects of different PEO molecular chains on biological response were discussed.     

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 5s and 24h. 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 32min and 24h 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.