Biomedical subjects

C H Gemmell

Publications and source records attributed to C H Gemmell.

At least 19 recordsLinked to original sources

PubMed2001

Platelet interactions with titanium: modulation of platelet activity by surface topography.

Endosseous implants initially come into contact with blood. Thus, the nature of the interactions between blood and implanted endosseous implants may influence subsequent bone healing events in the peri-implant healing compartment. We conducted studies to address the following question: Does implant surface microtexture modulate platelet activity? We used commercially pure Ti (cpTi) disks with four different surface finishes: dual acid-etched (DAE), 320 grit (320G) abraded, machined, and p1200 polished cpTi. Surfaces were characterized by scanning electron microscopy (SEM) and optical profilometry. The DAE and 320G surfaces presented more complex microtextures than the machined or polished surfaces. Platelet activities were measured by quantifying platelet adherence, platelet-derived microparticle (MP) formation, and P-selectin expression as function of surface type. Platelet adhesion, measured using a lactate dehydrogenase (LDH) assay. was increased on DAE and 320G surfaces compared to machined and polished surfaces (p < 0.05). M P formation and P-selectin expression, assayed by flow cytometry, also showed increased activation of platelets on DAE and 320G surfaces. Because increased activation of platelets may lead to up-regulation of osteogenic responses during bone healing, these results may explain the enhanced osteoconductivity known to occur with DAE cpTi surfaces in comparison with machined cpTi surfaces.

Blood Platelets↗

PubMed2001

Activation of platelets by in vitro whole blood contact with materials: increases in microparticle, procoagulant activity, and soluble P-selectin blood levels.

Non-adherent platelets and plasma were analyzed for evidence of platelet activation after whole blood contact with materials under conditions of low shear for one hour at 37 degrees C. The contact involved adding heparinized whole blood to small diameter tubes that were connected to two arms extending from a rocking platform. For all surfaces (polyethylene, polypropylene, Silastic, PVA hydrogel) tested there was strong evidence of platelet activation in the bulk blood: platelet-derived microparticles, procoagulant platelet membranes and soluble P-selectin levels. Flow cytometric quantification of microparticles (MPs) was highly sensitive and entailed the direct determination of microparticle concentrations as opposed to the traditional quantification of microparticle percentages (relative to total number of MPs and platelets). Whole blood contact with polypropylene surfaces led to the greatest drops in bulk platelet counts and also to the lowest increases in microparticle concentrations. Flow cytometry was also used to assess procoagulant levels (annexin V binding) within a light scatter region known to contain platelets and some large microparticles. All surfaces were noted to generate a significant procoagulant population that was, based on forward light scatter, mostly very small platelets or large microparticles. In contrast, most of the P-selectin positive platelets were averaged sized. Lastly. all surfaces generated soluble P-selectin levels that were approximately double the level (25 ng ml(-1)) noted in the resting whole blood samples. In addition to our previous reports, these findings support the observation that there is strong evidence of platelet activation in the bulk that we anticipate will ultimately lead to more relevant in vitro testing of the compatibility of platelets towards materials.

Biocompatible Materials↗

PubMed2000

What really is blood compatibility?

The criteria for nonthrombogenicity are classically defined as long clotting times and minimal platelet deposition. The inability to point to unequivocal progress in the development of truly nonthrombogenic materials, highlights the inadequacy if not actually invalidity of these criteria. Our approach is to define nonthrombogenicity in terms of: (1) a thrombin production rate constant, kp < 10(-4) cm s(-1); (2) low platelet consumption and low degree of platelet activation (e.g., microparticle formation); (3) perhaps some platelet spreading; and (4) low complement and leukocyte activation. Only when the target becomes clear, will it be possible to identify clear strategies for producing the materials we need.

Animals↗

PubMed2000

Flow cytometric evaluation of material-induced platelet and complement activation.

Flow cytometry is used to characterize the activation state of platelets and leukocytes within whole blood after contact for 4 h at 37 degrees C with various materials under conditions of low shear. The contact involved adding heparinized whole blood to small diameter tubes that were connected to two arms extending from a rocking platform. For all surfaces (polyethylene, polypropylene, Silastic, PVA hydrogel) tested there was strong evidence of platelet activation in the bulk blood: platelet-derived microparticles. P-selectin expression and platelet-leukocyte aggregates. Only contact with PVA hydrogel surfaces led to dramatic increases in CD11b up-regulation on monocytes and neutrophils that was inhibited by complement inhibition (sCRI). Flow cytometry was also used to evaluate the effectiveness of various agents to inhibit material-induced complement activation. The assay involved incubating 10 microm polystyrene beads for 1 h with serum at 37 degrees C before isolating the beads so as to label them with a monoclonal antibody against a neoantigen on SC5b-9. The beads were then identified by flow cytometry and the fluorescence associated with their SC5b-9 level recorded. The ability of C1-INH, pentamidine and benzamidine to moderately inhibit SC5b-9 levels suggests a role for classical complement activation in material-induced complement activation.

Biocompatible Materials↗

PubMed1999

The fibrinogen gamma chain dodecapeptide inhibits agonist-induced aggregation of rabbit platelets and fibrinogen binding to rabbit glycoprotein IIb-IIIa.

The HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the y chains and the RGD sequences in the Aalpha chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen gamma chain sequence. Proteins of approximately 135 kDa and approximately 95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.

Animals↗

PubMed1998

Assessment of material-induced procoagulant activity by a modified Russell viper venom coagulation time test.

Platelet activation is an inevitable consequence of blood-material interactions. The ability of those activated platelets and platelet-derived microparticles to enhance coagulation reactions leading to thrombin/fibrin formation has not been well studied despite its potential significance. Activated platelets and platelet-derived microparticles are known to dramatically enhance the catalytic efficiencies of the tenase and prothrombinase complexes. In this paper, a modified Russell viper venom coagulation time test is used to quantitate material-induced procoagulant activity due to the generation of activated phospholipid surfaces. In our test system, polyethylene and Silastic tubes were filled with heparinized whole blood and left to gently flow back and forth at 37 degrees C. After 1 h, the blood within the tubes was gravity drained and the plasma fraction assayed for procoagulant activity. The clotting times were determined by a Coag-A-Mate X2 instrument after the automated addition of Russell viper venom (to activate factors V and X) and calcium ions. Appreciable procoagulant activity was generated during whole blood contact within polyethylene and Silastic tubes although significantly greater activity was generated by the latter surface. As previously reported, platelet-derived microparticles also were detected by flow cytometry. Filtration of the plasma after material contact through a 0.1 microm filter led to substantial gains in clotting times and to near complete removal of microparticles, indicating that the material-induced microparticles likely were responsible for the procoagulant activity.

Factor V↗

PubMed1998

Platelet adhesion onto artificial surfaces: inhibition by benzamidine, pentamidine, and pyridoxal-5-phosphate as demonstrated by flow cytometric quantification of platelet adhesion to microspheres.

An appreciable effort is directed toward designing strategies to minimize platelet interactions with artificial surfaces, because their reactivity is thought to promote thrombus formation and lead to materials failure. Although platelet glycoprotein Ib/IX (GPIb/IX) and glycoprotein IIb/IIa (GPIIb/IIIa) receptors are thought to mediate adhesion, whether GPIIb/IIIa receptors are activated and how this might occur are largely unknown and are the focus of this article. There are a few ways, other than thrombin generation, that blood contact with artificial surfaces can lead to GPIIb/IIIa activation. Complement activation can lead to products capable of activating platelets (C1q, C5b-9), and contact between platelet CD32 (FcgammaRII) receptors and immobilized immunoglobulin G could also activate platelets. In this article the potential role of these processes was evaluated by using various inhibitors in a microsphere-based platelet adhesion immunoassay. Polystyrene microspheres (10 microm) were incubated in platelet-rich plasma before flow cytometric analysis of beads for adherent platelets. The data eliminated occupancy of the FcgammaRII receptor (by use of IV.3 blocking antibody), C5b-9 production (by use of sCR1), and the indirect action of factor XIIa on complement components (by use of corn trypsin inhibitor) as playing roles in supporting platelet adhesion. Agents directed against the first complement component (benzamidine, pentamidine, pyridoxal-5-phosphate) were effective inhibitors of platelet adhesion and were also demonstrated to inhibit SC5b-9 and C3d levels on the bead surface after serum incubations. Because these agents are not highly specific, it can not be concluded that C1q is a mediator of adhesion. These agents were also demonstrated to inhibit fluorescein isothiocyanate-fibrinogen binding to activated washed platelets, therefore indicating that fibrinogen receptor expression is a requirement for platelet adhesion.

Benzamidines↗

PubMed1998

Ethanol inhibits thrombin-induced secretion of the contents of human platelet dense and alpha-granules and lysosomes.

Moderate consumption of alcoholic beverages is associated with a reduction in thromboembolic complications of coronary artery disease, possibly partially attributable to inhibition by ethanol of platelet responses to some aggregating agents. Although ethanol is known to inhibit thrombin-induced secretion of platelet dense granule contents, the effect of ethanol on secretion of alpha-granule and lysosomal contents has not been studied. Using suspensions of washed platelets, and a range of thrombin concentrations (up to 0.1 U/ml), we examined the effect of 87 mM ethanol on secretion of [14C]serotonin from prelabelled platelets as a measure of secretion of dense granule contents. Secretion of alpha-granule and lysosomal contents was examined by flow cytometric measurement of the surface expression of CD62P (P-selectin) and CD63, respectively. Secretion of the lysosomal enzyme, beta-N-acetylglucosaminidase was also quantified. Results were expressed as % of maximum response induced by 1 U/ml thrombin. Ethanol inhibited the thrombin-induced secretion of both dense and alpha-granule contents (P <0.001, 2-way ANOVA), and of lysosomal contents (P <0.005 for CD63 expression and P <0.001 for beta-N-acetylglucosaminidase secretion). When platelets were pretreated with aspirin, thrombin-induced secretion of storage granule and lysosomal contents was slightly inhibited, but secretion was inhibited by ethanol to the same extent as the untreated platelets, indicating that this inhibition was independent of thromboxane A2. Surface expression of CD63 occurred at lower thrombin concentrations than those required for secretion of beta-N-acetylglucosaminidase, possibly due to the presence of some CD63 on granule membranes. Although the role of lysosomal contents in thrombus formation is not established, some constituents of storage granules are known to augment thrombus formation; ethanol's inhibition of their secretion by stimulated platelets may contribute to its beneficial effect on thromboembolism.

Blood Platelets↗

PubMed1997

A flow cytometric immunoassay to quantify adsorption of complement activation products (iC3b, C3d, SC5b-9) on artificial surfaces.

Crosslinked agarose microspheres and various polystyrene microspheres were analyzed for complement components after incubation with serum at 37 degrees C for times up to 2 h. Quantification involved direct flow cytometric analysis of the beads after the bound complement proteins were indirectly fluorescently tagged by use of a monoclonal antibody against a complement protein: C5b-9, iC3b, C3d, C4d, Bb, C3a, and C1q. Calibration with fluorescein microbead standards demonstrated that the membrane attack complex (SC5b-9) was surface bound on all surfaces and that the surface concentration gradually increased to levels as high as 0.5 micrograms/cm2. Further, the surface bound represented a substantial percentage of the total generated. The iC3b level on polystyrene beads rapidly reached 0.09 micrograms/cm2 and the C3d levels were an order of magnitude less. On agarose beads the iC3b levels continually rose to 0.17 micrograms/cm2 and, as before, the C3d levels were substantially lower. The surface concentration of C4d and Bb on both surfaces were significant but less than 1.0 ng/cm2. There was minimal evidence of C3a and C1q adsorption for any surface. Use of amino-polystyrene beads moderately reduced the level of bound iC3b, C3d, and SC5b-9, whereas carboxylated beads reduced the levels by almost a factor of two. The appreciable amounts of iC3b and SC5b-9 consistently noted on the artificial surfaces tested in this paper suggests that for these two activation products in vitro analysis of material induced complement activation should also include surface analysis.

Adsorption↗

PubMed1997

Flow cytometric analysis of material-induced platelet activation in a canine model: elevated microparticle levels and reduced platelet life span.

Assessment of material-induced platelet activation is important given that it is thought to be a major mechanism of biomaterials thrombogenicity. We monitored, by flow cytometry, platelet microparticle (MP) levels in the circulation during the connection of polyvinyl alcohol (PVA) hydrogel and polyethylene (PE) test segments (3.18 mm ID, 20 and 50 cm L) to our chronically shunted beagle dogs. We report that circulating microparticle levels were dependent on test segment material, length, and time. The connection of 50-cm lengths of PVA hydrogel test segments led to MP levels two to three times greater than background at 48 h, while the connection of polyethylene test segments did not lead to elevated microparticle levels. MP levels were near background 24 h after removal of the PVA test segment. To determine platelet life span during the connection of test segments, platelets were labeled in vivo with biotin and their disappearance monitored flow cytometrically. While platelet life span for shunted dogs (no test segment) was 4.7 +/- 0.2 days, the connection of PVA hydrogel test segments led to a platelet life span of < 2 days.

Animals↗

PubMed1996

Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: direct measurement of percent release of beta-thromboglobulin and assessment by flow cytometry of P-selectin expression.

Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of alpha-granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP-induced aggregation in citrated platelet-rich plasma (PRP). Measurements were: percent release of 14C-serotonin from prelabeled platelets, radio-immunoassay of beta-thromboglobulin (beta TG), and expression on the platelet surface of the alpha-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 +/- 8.3% of beta TG and 54.1 +/- 4.6% of 14C-serotonin were released (mean +/- SEM, n = 13); aspirin treatment reduced these values to 6.0 +/- 1.2 and 1.0 +/- 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 +/- 1.7% release of beta TG and 2.1 +/- 0.4% release of 14C-serotonin; these low values were not appreciably affected by aspirin. During ADP-induced primary aggregation in PRP anticoagulated with FPRCH2CI (PPACK), only 4.7 +/- 0.9% release of beta TG and no detectable release of 14C-serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 microM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2-10 microM), the mean fluorescence intensity due to P-selectin was < 14% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P-selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2+ concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P-selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both alpha-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP.

Adenosine Diphosphate↗

PubMed1996

Material-induced up-regulation of leukocyte CD11b during whole blood contact: material differences and a role for complement.

Material-induced thrombogenicity is in part a consequence of leukocyte activation. To evaluate and compare material-induced platelet damage, we have expanded our in vitro flow cytometric immunoassay to include assessment of leukocyte activation. We have used a very simple system whereby fresh, heparinized whole blood contacts materials for 1 h at 37 degrees C under low shear. Unlike other tests that focus on adherent leukocytes, this assay evaluates the leukocytes in the whole blood drained from the tube (1.57 mm internal diameter, 25 cm length) after material contact. We demonstrate that whole blood contact with a polyvinyl alcohol (PVA) hydrogel surface leads to a twofold up-regulation in CD11b surface expression of all monocytes and neutrophils. The activation is metal-ion dependent and highly material dependent in that blood contact with polyethylene and Silastic surface leads to minimal activation. The shedding of L-selection as a marker of leukocyte activation was found to be unsuitable in our assay given it ease of shedding in resting heparinized whole blood. Further, plasma levels of complement components Bb and sC5b-9 (ELISA assays) were significantly elevated only after blood contact with PVA hydrogel surfaces (9.4 micrograms/mL sC5b-9 and 9.6 micrograms/mL Bb). Use of recombinant soluble human CR1 (sCR1) to inhibit the action of the C3 and C5 convertases completely inhibited sC5b-9 levels in whole blood after contact with PVA hydrogel surfaces and inhibited CD11b up-regulation by over 70%, suggesting that material-induced leukocyte activation is partially mediated by C5a production.

Biocompatible Materials↗

PubMed1995

Platelet activation in whole blood by artificial surfaces: identification of platelet-derived microparticles and activated platelet binding to leukocytes as material-induced activation events.

Because the lack of thromboresistant vascular biomaterials is in part due to platelet activation, we have attempted, by using fluorescence-activated flow cytometry, to fully characterize the platelet population after in vitro material contact with whole blood. We have used a very simple, near-physiologic system whereby whole blood, anticoagulated with D-phenylalanyl-L-prolyl-arginyl chloromethyl ketone (thrombin inhibitor), contacts materials for 1 hour at 37 degrees C, under low shear. Unlike other tests of platelet compatibility that focus on adherent platelets, this assay evaluates the platelets in the whole blood drained from the tube (1.57 mm internal diameter, 25 cm length) after material contact. We demonstrate for the first time significant materials-induced microparticle formation. One-hour contact with Silastic, polyethylene, and polyvinyl alcohol hydrogel surfaces lead to 30 +/- 1, 33 +/- 4, and 43 +/- 4 x 10(9) microparticles/L, respectively, whereas resting blood samples contained only 10 +/- 1 x 10(9) microparticles/L. In addition, significant increases in activated platelet(s) binding to neutrophils/monocytes after material contact were noted for all surfaces tested. For polyvinyl alcohol hydrogel surfaces a greater than 500% increase in the fluorescent intensity over that of resting whole blood was attained. The addition of monoclonal antibodies to GPIIb/IIIa (A2A9), the tetrapeptide adhesion ligand RGDS (arginine-glycine-aspartate-serine), or the calcium ion chelator ethyleneglycol-bis-(B-aminoethyl-ether)- N,N,N',N'-tetraacetic acid to the whole blood before material contact fully inhibited all platelet reactivity noted for all surfaces--platelet microparticles, platelet P-selectin expression, loss of platelets from bulk, and the formation of platelet/leukocyte aggregates--thereby indicating that material-induced platelet activation is a calcium-dependent process involving GPIIb/IIIa receptors.

Antibodies↗

PubMed1995

Elevated platelet-derived microparticle levels during unstable angina.

OBJECTIVE: To use fluorescent-activated flow cytometry coupled with activation-dependent and -independent platelet-specific monoclonal antibodies in a pilot study to assess the degree and time course of platelet activation events in patients presenting within 24 h of onset of Canadian Cardiovascular Society class 4 angina. BACKGROUND: Although activated platelets play a key role in the pathogenesis of unstable angina, the development of simple assays to quantify platelet activation events directly is lacking. METHODS: Blood samples were drawn from six unstable angina patients from the coronary care every 4 h over a 24 h period into a fixative and analyzed the following day. All patients were on acetylsalicylic acid and heparin. Comparisons were made with six healthy, medication-free volunteers. Platelets were defined by flow cytometry as positive for fluorochrome-labelled monoclonal antibody to glycoprotein Ib (AP1) and within the single intact platelet window defined by scatter characteristics. The presence of the fluorochrome-labelled activation-specific monoclonal antibody (KC4.1 for anti-P-selectin, PAC-1 for activated glycoprotein IIb/IIIa) was used to determine the percentage of activated platelets. Platelet activation-dependent microparticles were identified by gating on AP1-positive events and defining microparticles (percentage of total platelet events) as being smaller (forward size scatter) than single intact platelets. RESULTS: There was a marked, sixfold increase in microparticle generation (17 +/- 7% versus 2.8 +/- 1.4%) in the unstable angina patients (P = 0.001) compared with healthy volunteers. Further assessment of six coronary care unit patients with nonischemic cardiac disorders demonstrated a highly variable intermediate level of microparticle generation (11 +/- 7%). No differences in activated glycoprotein IIb/IIIa expression were noted for the various groups and P-selectin expression was lower in the unstable angina patients (6 +/- 2% versus 12 +/- 3%, (P = 0.007). CONCLUSIONS: This pilot study suggests that measuring circulating platelet microparticle levels is a simple yet useful parameter for the assessment of platelet activation in unstable angina compared with activation markers on intact whole platelets. Despite antiplatelet and antithrombin therapy, significant platelet activation occurred in these patients over the initial 24 h. Flow cytometry may be a useful tool in assessing the efficacy of newer therapeutic modalities.

Adult↗

PubMed1993

Platelet-derived microparticle formation involves glycoprotein IIb-IIIa. Inhibition by RGDS and a Glanzmann's thrombasthenia defect.

While the physiologic role of platelet microparticles may include a stable, physical dispersion of concentrated surface procoagulant activity the mechanism(s) of platelet vesiculation remains unknown. We demonstrate using flow cytometric methods a central role for the beta 3 integrin glycoprotein (GP) IIb-IIIa complex and its ligand tetrapeptide Arg-Gly-Asp-Ser (RGDS) binding site in platelet vesiculation. Time- and calcium-dependent vesiculation of platelets in response to ADP, collagen, thrombin, phorbol myristate acetate, and the thrombin peptide SFLLRN were dramatically inhibited, in a concentration-dependent manner, by monoclonal antibodies to GPIIb-IIIa (A2A9, 7E3, PAC1) and RGDS. Complete inhibition with A2A9 and RGDS occurred at 7.5 micrograms/ml and 75 microM, respectively, while control antibodies and a mock peptide had no effect. Platelet vesiculation requires intact GPIIb-IIIa and is fully supported by the intracellular pool of GPIIb-IIIa alone since de-complexing of this heterodimer by calcium chelation completely abolished microparticle formation in response to collagen (no alpha-granule release) but not to thrombin or SFLLRN. A central role for GPIIb-IIIa is supported by the near total inability of Glanzmann's thrombasthenic (type I) platelets to vesiculate in response to thrombin, ADP, collagen, and phorbol 12-myristate 13-acetate. This extends the biologic roles of GPIIb-IIIa to include platelet vesiculation and suggests that one or all of its binding ligands play a role.

Amino Acid Sequence↗

PubMed1991

Factors affecting the interaction of tissue factor/factor VII with factor X in a heterogeneous tubular reactor.

A novel reactor recently described for studying phospholipid-dependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further characterized. The reactor is a capillary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s-1 to 1,200 s-1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, Spectrozyme TMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

Calcium↗

PubMed1990

Utilization of a continuous flow reactor to study the lipoprotein-associated coagulation inhibitor (LACI) that inhibits tissue factor.

A microperfusion system containing a glass capillary, the inner surface of which is coated with a phospholipid bilayer containing tissue factor, was used to explore the requirement for factors VIIa and Xa in the complex formed with the lipoprotein-associated coagulation inhibitor (LACI). Various combinations of factors VIIa, Xa, and LACI were perfused together or sequentially at a wall shear rate of 300 sec-1; a final perfusion of factors X and VIIa was performed to evaluate the residual tissue factor activity. Factor Xa concentration at the outlet of the tube was determined using a chromogenic substrate. In the presence of factors VIIa, Xa, and LACI, complete inhibition of tissue factor was observed on both phosphatidylcholine (neutral surfaces) and on phosphatidylserine/phosphatidylcholine (acidic) surfaces; omission of factors Xa or LACI resulted in no inhibition. The absence of factor VIIa in the initial perfusion steps resulted in no inhibition on neutral surfaces whereas about 90% inhibition was observed on acidic surfaces. Initial perfusion with factor Xa, but not LACI, followed by the remaining protein components, resulted in an inhibitory complex. Thus, it appears that a tissue factor:factor Xa:LACI complex can form in the absence of factor VIIa on acidic surfaces; moreover, our data imply a tissue factor binding site for factor Xa, but not for LACI.

Calcium↗

PubMed1990

The effects of shear rate on the enzymatic activity of the tissue factor-factor VIIa complex.

A novel enzyme reactor for studying phospholipid-dependent reactions was used to explore the effects of flow on tissue factor (TF)-initiated coagulation. Capillary tubes (0.27 mm i.d.) were coated with a phospholipid bilayer containing TF, a transmembrane protein that is an essential cofactor for factor VII. Production of factor Xa exiting the tube was monitored with time during perfusion of the capillary with factor X (50 to 1500 nM) in the presence of factor VIIa (10 nM). Steady-state production of factor Xa as a function of [FX] was determined by chromogenic assay (Spectrozyme Xa) for a range of wall shear rates (25 to 3000 sec-1). Diffusion was found to play a major limiting role in FXa production for TF:30% phosphatidylserine (PS)/70% phosphatidylcholine (PC) surfaces. In contrast, TF/PC surfaces slowed the reaction sufficiently to enter a kinetically controlled regime where shear fluid had little effect on Km. In contrast with classical enzyme kinetic theory there was a three-fold increase in Vmax as shear increased from 25 to 300 sec-1. This finding implies a direct effect of shear on the kinetics of factor X activation by TF/FVIIa. The perfusion system is simple to use and offers the potential for studying the role of flow on a wide variety of enzymatic reactions related to coagulation.

Blood Flow Velocity↗