Cynthia H. Gemmell

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.

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.

Does surface chemistry affect thrombogenicity of surface modified polymers

With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, and cell deposition, by scanning electron microscopy after blood exposure and clotting times among a group of 12 unmodified and plasma modified tubings. All materials activated platelets and leukocytes to detectable levels, although some materials increased the value of one activation parameter but not another. Unmodified materials [polyethylene (PE), Pellethane (PEU), latex, nylon, and Silastic] and modified materials (H(2)O plasma treated PE and PEU, CF(4) plasma treated PE, fluorinated PEU, NH(4) plasma treated PEU, polyethylene imine treated PEU, and heparin treated PEU) were characterised by XPS and contact angle. The objective of this project was to define a series of assays for the evaluation of hemocompatibility of cardiovascular devices with a view to clarify the specific requirements of ISO-10993-4, and to define an appropriate screening program for new blood contacting biomaterials. PE, PE--CF(4), PE--H(2)0, PEU--F, latex, and PEU-heparin were the exceptions to the general observations, although each behaved differently. PE proved to be least reactive, whereas PE-CF(4) was most reactive by several assays. Platelet microparticle formation (determined by flow cytometry), PTT, postblood exposure SEM, total SC5b-9, C3a, and platelet and leukocyte loss (cell counts) were able to distinguish differences among these materials, and often, but not always, showed expected correlations.

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.

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.

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°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.

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 β‐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 α‐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 β‐thromboglobulin (βTG), and expression on the platelet surface of the α‐granule constituent, P‐selectin, by flow cytometry. During the second phase of ADP‐induced aggregation, 69.0 ± 8.3% of βTG and 54.1 ± 4.6% of 14C‐serotonin were released (means ± 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 βTG 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 FPRCH2Cl (PPACK), only 4.7 ± 0.9% release of βTG 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 μM), 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 μM), 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 α‐granule and dense granule contents during ADP‐induced secondary aggregation in citrated PRP.

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.

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.

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.