polymerization
7.1.1 IntroductionFactor XIII (FXIII) is a coagulation protein that plays an essential role in physiologic
hemostasis where it can regulate fibrinolysis[141], wound healing[142], and
angiogenesis[143]. Clinically, FXIII deficiency results in hemorrhage and impaired wound
healing[144]. FXIII is a plasma protransglutaminase composed of 2 A (FXIII-A) and 2 B (FXIII-B) subunits that circulate as a heterotetrameric zymogen (FXIII-A2B2)[145]. FXIII
activation occurs when thrombin cleaves an N-terminal activation peptide from FXIII-A, and calcium mediates the dissociation of the inhibitory FXIII-B subunits, producing
FXIIIa[145]. FXIIIa confers clot stability by cross-linking fibrin fibers in the fibrin matrix, thus altering its rheologic properties. FXIII originates from a family of transglutaminases
(TG), TG are known to catalyze the formation of covalent ε-(γ-glutamyl) lysyl bond where a lysine ε-amino group is cross-linked to the glutamine of a γ-carboxymide group[141].
FXIIIa can also cross-link inhibitors of fibrinolysis to fibrin, further enhancing fibrin’s insolubility and plasmin resistance. Inhibitors cross-linked to fibrin include α2-
antiplasmin (α2AP), thrombin activatable fibrinolysis inhibitor (TAFI), and plasminogen
activator inhibitor-2. In all, there are twenty-five FXIIIa substrates that can be divided into
five categories: coagulation factors, components of the fibrinolytic system, adhesive and extracellular matrix proteins, intracellular cytoskeletal proteins, and others[145]. Previous
123 demonstrated by either the rapid lysis of clots formed in the absence of α2AP or when α2AP
activity is neutralized[146]. Moreover, in addition to being present in plasma, FXIII-A is also
present in platelets in large quantities[147–149]. The concentration of FXIII-A is 100X greater in the platelet cytoplasm than in plasma[150]. Recently, Mitchell et al. have shown
that activated platelets externalize this pool of FXIII-A onto their membranes and that this
pool is functional in cross-linking α2AP and confers lytic resistance in platelet rich thrombi
under flow[151].
The (patho)physiologic roles for FXIIIa activity derived from either platelet FXIII-A (cFXIII) or plasma FXIIIa (FXIIIa) are still not well-understood. Previous research has
shown that human and murine whole blood clots formed in the absence of plasma FXIII or in the presence of a small molecular inhibitor for FXIIIa have reduced red blood cell
retention and are significantly smaller than control clots[9]. Further work proved that FXIIIa mediates red blood cell retention in clots by cross-linking fibrin alpha chains[152]. Using
microfluidic hemostasis models and whole blood perfusion under flow we aimed to delineate the ability of cFXIII and FXIIIa to confer platelet-rich thrombus stability independent of
polymerized fibrin.
7.1.2 Materials and Methods 7.1.2.1 Materials
The following reagents were stored according to manufacturer’s instructions: Corn Trypsin Inhibitor (CTI, Haematologic Technologies, Essex Junction, VT), phycoerythrin
124 Franklin Lakes, NJ). Fibrinogen from human plasma, Alexa Fluor 647 conjugate (Invitrogen Life Technologies, Carlsbad, CA). Equine fibrillar collagen type I (Chronopar, Chronolog,
Havertown, PA), Dade Innovin (Siemens Healthcare USA, Malvern, PA), 1,3,4,5- Tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (T101) (Zedira, Darmstadt,
Germany), Gly-Pro-Arg Pro (Billerica, Massachusetts, EMD Millipore), Sylgard 184 Silicone
Elastomer Kit (Sylgard, Ellsworth Adhesives, Germantown, WI), Iodacetamide (Sigma Aldrich, St. Louis, MO).
7.1.2.2 Blood Collection and preparation
Phlebotomy was approved by the University of Pennsylvania Institutional Review
Board and performed on consenting donors in accordance with the Declaration of Helsinki. Donors abstained from alcohol 48 h prior to blood donation and were free of oral
medication for a minimum of 7 days prior to donation.
7.1.2.3 Microfluidic Assay
Whole blood was taken from healthy donors using standard phlebotomy techniques. Blood was drawn into 40 µg/ml CTI or FPR-chloromethylketone (PPACK, Haematologic
Technologies, Essex Junction, VT, 100 µM final concentration). CTI inhibits the contact pathway for studies of surface-triggered coagulation while PPACK inhibits thrombin
generation in order to examine platelet function in the absence of thrombin in vitro. Blood samples were treated with 0.125 µg/ml fluorescently conjugated anti-CD61a antibody to
label platelets and fluorescently conjugated fibrinogen to label fibrin(ogen) 5 min prior to initiation of flow assays. All healthy donor microfluidic experiments were completed within
125 Microfluidic fabrication methods and device specifications were previously described [24,33,34]. Microfluidic channels ran perpendicularly over a 250 µm wide strip of patterned
equine fibrillar collagen type I (Chronopar, Chronolog) or Tissue Factor (TF) bearing collagen type I surfaces (Dade Innovin, Siemens Healthcare USA, Malvern, PA).
Epifluorescent microscopy and image acquisition were performed in real-time as previously
described [23,24].
Blood samples were perfused at an initial venous wall shear rate of 200 s-1 or an
initial arterial wall shear rate of 800 s-1 in a previously designed pressure relief mode for 20
min [37]. Platelet and fibrin accumulation were analyzed as previously described [24].
7.1.3 Results
7.1.3.1 Nonspecific FXIIIa inhibitor confers gross platelet instability in the presence of GPRP at constant flow conditions
To investigate the fibrin-independent role of FXIIIa activity and function, non- selective FXIIIa inhibitor iodoacetamide was used ex vivo in the presence or absence of
GPRP, a short four peptide sequence that inhibits fibrin polymerization. Following incubation with iodoacetamide and GPRP, whole blood was perfused over a TF/collagen
surface under venous shear rates. Decreases in platelet fluorescence intensity were observed over time in the absence of GPRP indicating a thrombin dependent role of FXIIIa (Figure
7-1A). The addition of exogenous iodoacetamide had not effect of fibrin initiation and accumulation over time (Figure 7-1B). A combination of GPRP and iodoacetamide resulted
126 fluorescence over time (Figure 7-1B; red line). Exogenous GPRP completely inhibited fibrin generation under flow independent of iodoacetamide consistent with previous studies
under flow (Figure 7-1B) [37].
Figure 7-1 Inhibition of FXIIIa in the presence or absence of fibrin polymerization (±GPRP) on TF/collagen surfaces at 200 s-1.
(A & B), Platelet deposition dynamics measured by platelet fluorescence in the presence or absence of GPRP (5 mM). (C & D), Fibrin deposition dynamics measured by fibrin fluorescence in the presence or absence of GPRP (5 mM). Lines with shaded traces and points are mean and standard deviation of 4 clotting events measured in 45 sec intervals over 10 min for one healthy donor.
127 7.1.4 Discussion
Here we demonstrate for the first time a potential role for FXIIIa stability of platelet
rich thrombi under flow in the absence of fibrin. Further work must be done to elucidate the mechanisms by which FXIIIa can stabilize platelet rich clots. It is unclear whether the
weakened clot architecture and thrombus disintegration is due to the cellular form of FXIIIa
(cFXIIIa) or the plasma-derived form (FXIIIa). More specific FXIIIa inhibitors with micro- molar affinity such as 1,3,4,5-Tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (T101)
should be tested ex vivo. T101 can be used exogenously in the presence or absence of thrombin inhibitors over collagen or collagen/TF surfaces. Pressure relief conditions can be
used to build occlusive clots under flow for a more rigorous test of platelet/fibrin clot strength and stability. To further elucidate the role of cFXIIIa, GR145503 can be used to
abate platelet aggregation under flow in order to study platelet GPVI and collagen interactions. Finally, fluorescent amine donor substrates can be used to detect
transglutaminase activity under flow to delineate the contributions of FXIIIa activity emanating from platelets or platelet-bound fibrin(ogen).
128