Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces

Abstract

CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of ‘self-recognition’. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47-modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils.

The use of CD47-modified biomaterials to mitigate the immune response

“…63 Using an ex vivo model system, which perfuses whole blood over CD47 immobilized or unmodified control surfaces, we showed that platelet activation was significantly inhibited as a function of CD47 exposure. 63 Specifically, we showed that the expression of the protein surface marker of platelet activation, P-selectin (CD62P), was significantly reduced after 3 h exposure to CD47-modified blood conduits. 63 Scanning electron microscopy demonstrated that the platelet attachment was markedly reduced on CD47-modified surfaces compared with unmodified control surfaces.…”

The Use of the <em>Ex Vivo</em> Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

“…Reducing the covalently appended PDT groups with tris(2-carboxyethyl) phosphine hydrochloride (TCEP) 11 yields a thiolated surface that can be subsequently reacted with therapeutic moieties. Detailed herein and previously 12,13 , recCD47, further modified with the addition of a C-terminal poly-lysine tail 12,13 , is reacted with Sulfosuccinimidyl-4-[Nmaleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) for 1 hr to generate thiol-reactive groups, allowing for a monosulfide bond formation between the tubing and recCD47 11 . The anti-inflammatory capacity of the CD47 functionalized surfaces was tested, ex vivo, using the Chandler Loop Apparatus with whole human blood, which was originally described in 1958 as an in vitro model of thrombotic coagulation 15 .…”

“…Signal-regulatory protein alpha (SIRPα), the cognate receptor for CD47, and a member of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing family of transmembrane proteins, is expressed on cells of myeloid origin 14 . We have previously demonstrated that CD47, via SIRPα-mediated cell signaling, down-regulates the immune responses to polyurethane (PU) and polyvinylchloride (PVC) in in vitro, ex vivo, and in vivo models [11][12][13] . Central to our investigations is a relatively novel photoactivation chemistry, described herein, in which chemically reactive thiol groups are covalently appended to polymeric tubing by reacting the tubing with a multifunctional polymer (PDT-BzPh), composed of 2-pyridyldithio (PDT), the photoreactive benzophenone (BzPh) and a carboxy-modified polyallylamine [11][12][13] .…”

“…We have previously demonstrated that CD47, via SIRPα-mediated cell signaling, down-regulates the immune responses to polyurethane (PU) and polyvinylchloride (PVC) in in vitro, ex vivo, and in vivo models [11][12][13] . Central to our investigations is a relatively novel photoactivation chemistry, described herein, in which chemically reactive thiol groups are covalently appended to polymeric tubing by reacting the tubing with a multifunctional polymer (PDT-BzPh), composed of 2-pyridyldithio (PDT), the photoreactive benzophenone (BzPh) and a carboxy-modified polyallylamine [11][12][13] . Reducing the covalently appended PDT groups with tris(2-carboxyethyl) phosphine hydrochloride (TCEP) 11 yields a thiolated surface that can be subsequently reacted with therapeutic moieties.…”

“…Research in our laboratory has focused on appending recombinant CD47 (recCD47) to polymeric biomaterials as a strategy to mitigate the FBR and increase the efficacy of these materials. CD47 is a ubiquitously expressed transmembrane protein with a known role in immune evasion, conferring "self" status upon expressing cells [7][8][9][10] and shows promise at conferring biocompatibility when appended to polymeric surfaces [11][12][13] . Signal-regulatory protein alpha (SIRPα), the cognate receptor for CD47, and a member of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing family of transmembrane proteins, is expressed on cells of myeloid origin 14 .…”