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. 2013 Jun;11(6):1163-71.
doi: 10.1111/jth.12209.

C560Rβ3 Caused Platelet Integrin αII B β3 to Bind Fibrinogen Continuously, but Resulted in a Severe Bleeding Syndrome and Increased Murine Mortality

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Free PMC article

C560Rβ3 Caused Platelet Integrin αII B β3 to Bind Fibrinogen Continuously, but Resulted in a Severe Bleeding Syndrome and Increased Murine Mortality

J Fang et al. J Thromb Haemost. .
Free PMC article

Abstract

Background and objectives: β(3)-Deficient megakaryocytes were modified by human β(3)-lentivirus transduction and transplantation to express sufficient levels of a C560Rβ(3) amino acid substitution, for investigation of how an activated αII b β(3) conformation affects platelets in vivo in mice.

Patient/methods: As in our previous report of an R560β(3) mutation in a patient with Glanzmann thrombasthenia, R560β(3) murine platelets spontaneously bound antibody that only recognizes activated αII b β3 bound to its ligand, fibrinogen.

Results: With this murine model, we showed that αII b -R560β3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560β(3) 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560β(3) 'wild-type' mice remained alive. Pathologic examination revealed that R560β(3) mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560β(3) megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560β(3) washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo.

Conclusions: The results demonstrate that continuous occupancy of αIIb β3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state.

Keywords: glanzmann thrombasthenia; hematopoietic stem cells; integrin αIIbβ3; lentivirus gene transfer; megakaryocytes; platelets.

Conflict of interest statement

Disclosures: The authors have no conflict of interest to disclose.

Figures

Figure 1
Figure 1. C560β3 & R560β3 tx Recipient Platelets Expressed αIIbβ3
(A) β3-WPT Diagram. Viral 3'-long terminal repeat (LTR) enhancer/promoter was removed to self-inactivate vector (SIN) and αIIb gene promoter (nucleotides −889 to +35) directed megakaryocyte-specific transcription of cDNA with either nucleotide C1776T encoding a Cys (C) or Arg (R) at amino acid 560 of human β3. αIIb promoter binds GATA and Ets for high-level gene transcription in megakaryocytes while a repressor inhibits gene transcription in other lineages. The woodchuck hepatitis virus postregulatory element (WPRE) and central polypurine tract (cPPT) enhance transgene expression. (B) αIIbβ3 was Detected on Platelets Tranduced with C560Rβ3. Left, immunocytometric analysis showed the mean fluorescence intensity (MFI) for αIIb(+) platelets in C560Rβ3 mice (black) appeared at moderate levels compared to β3(−/− green; +/− grey; +/+ brown) controls. Right, only C560Rβ3 tx mice displayed detectable levels of human β3 on platelets (black) compared to MFI levels of platelet controls. Results represent 15 experiments analyzing platelets from β3(−/−,+/−,+/+) controls and 23 C560β3 mice and 20 R560β3. (C) αIIbR560β3 was Activated Continuously. Left, immunocytometric analysis revealed that C560β3 platelets (shaded peak) bound an Ab “D3” (recognizing an epitope exposed on high-affinity conformation of human β3 bound to ligand) only in the presence of a fibrinogen mimetic peptide containing Arg-Gly-Asp (+RGD). Right, in contrast, R560β3 platelets bound “D3” in the absence (−RGD) and presence (+RGD) of the peptide indicating that αIIbR560β3 was in an activated conformation. Negative control β3(−/−) platelets (green) failed to bind “D3”. Results represent four experiments using platelets from β3(−/−,+/−,+/+) controls and three C560β3 and R560β3 mice. (D) Immunocytometric Analysis of Fixed/Permeabilized Platelets. Fibrinogen was absent from β3(−/−) platelets, although β3(+/−;+/+) controls displayed appreciable levels of platelet fibrinogen. C560Rβ3 platelets bound, endocytosed, and stored fibrinogen (shaded peak). Results represent ten experiments using β3(−/−,+/−,+/+) controls and three C560β3 and R560β3 mice. (E) Immunocytometric Analysis Detected Activation of Platelets Treated with a mixture of ADP, epinephrine, and PAR4. The α-granule protein, P-selectin, was detected on the surface only after activation of C560β3 and R560β3 platelets (shaded peak) demonstrating that platelets expressing either form of β3 circulated normally in a quiescent manner. Results represent three experiments using platelets from β3(−/−,+/−,+/+) controls and three C560β3 and R560β3 mice.
Figure 2
Figure 2. R560β3 Mice had a Significantly High Mortality Rate
(A) Graph of Survival Rates for C560β3 and R560β3 tx Mice. Results demonstrate that 87% of C560β3 mice survived for at least 26 weeks post tx (n=23), while in contrast, 50% R560β3 animals died by 11 weeks after tx and only 35% of R560β3 mice survived until the experimental endpoint (n=20). Results with β3(−/−) mice tx with untransduced β3(−/−) bone marrow as a negative control (n=10) revealed that nearly 60% of β3(−/−) tx mice died by 1.5 weeks after lethal irradiation and bone marrow tx. Although, the endpoint survival of R560β3 mice was nearly identical to the 30% survival rate of β3(−/−) mice tx with untransduced marrow. (B) Tissue Analysis Revealed R560β3 Mice in Pathological Crisis. Top Left, shown is one of thirteen R560β3 mice that displayed pathological crisis, which usually resulted in death 48 hours after sudden onset of a hunched appearance and pale-white extremities. Top right, intact organs were isolated from R560β3 mice in pathological crisis. Analysis showed heart, lungs, brain, liver, and kidneys appeared normal in all mice, although gut appeared black and necrotic in one mouse which is consistent with results from hemocrlt counts indicating severe gastrointestinal bleeding. Tissues were fixed, sectioned, stained and analyzed as described in methods from nine R560β3 mice in crisis, nine R560β3 mice that appeared well, and three each of C560β3 and β3(−/−,+/−,+/+) controls. Bottom, R560β3 mice spleens (#1–5) appeared mostly abnormal in size and shape (large and/or necrotic) compared to a healthy spleen from a normal control (#6). Shown is spleen from five of nine R560β3 affected mice that underwent autopsy and tissue analysis. (C) R560β3 Mice had Abnormal Spleens. Top Left, H&E staining of spleen tissue indicated the C560β3 mice displayed normal spleen morphology at 200× magnification. In contrast (Top Right) extramedullary hematopoiesis (arrows) was detected in spleen sections from several R560β3 mice in crisis. Bottom Left, H&E staining indicated C560β3 mice displayed normal spleen morphology at 400× magnification. Bottom Right, in contrast numerous activated macrophages (arrows) were detected in the process of repair of pathological conditions within R560β3 murine spleens. (D) R560β3 Megakaryocytes were Shaped Abnormally within the Bone Marrow. Immunohistochemical brown staining with Ab recognizing integirn β3 showed megakaryocytes expressing C560Rβ3 in vivo (Magnification, 400×). Left panel shows that C560β3 megakaryocytes appeared normal in shape and size (arrow), while in contrast, the right panel revealed that R560β3 megakaryocytes appeared abnormal in shape and clustered within the bone marrow (arrow). Results represent observations of bone sections from β3(−/−,+/−,+/+) controls and three mice expressing either C560β3 or R560β3. (E) R560β3 Megakaryocytes Were in a Pathological State. As described in Part D, an Ab to integrin αIIb showed C560β3 megakaryocytes appeared healthy (top left arrow), while in contrast R560β3 Megakaryocytes appeared abnormal in shape (top right arrow). Interestingly, occasional R560β3 megakaryocytes had engulfed neutrophils by emperiopoiesis (bottom right arrow) indicating that the megakaryocytes were activated atypically. Result represents observations of bone sections from β3(−/−,+/−,+/+) controls and three C560β3 or R560β3 mice. (Magnification, 400×)
Figure 3
Figure 3. R560β3 Platelet Structure, Granular Content, and Function were Abnormal
(A) Light Microscopic Analysis of Wright/Giemsa Stained Peripheral Blood Smears. C560β3 platelets were normal in shape and size (left arrow). In contrast, R560β3 mice had some abnormally shaped platelets (right arrow). Results represent observations of six experiments using β3(−/−,+/−,+/+) controls and C560β3 or R560β3 tx mice. (Magnification 200×). (B) Electron Microscopy of Blood Platelets. Ultrathin sections were examined for platelet ultrastructure and granular distribution and content. High magnification images of C560β3 platelets displayed normal size, shape, and granule distribution. In contrast, R560β3 platelets were abnormally shaped with uneven granule and vacuole size and distribution (right arrows). A minimum of 100 sections were examined from each platelet sample. bar=1.0 µm. (C) Immunogold Labeling to Detect Platelet Fibrinogen by Electron Microscopy. Ultrathin sections of platelets were prepared and examined for surface and granular distribution of murine fibrinogen. Sections were placed on grids that were incubated with a rabbit Ab to murine fibrinogen and a goat anti-rabbit secondary antibody conjugated to (10nm) gold particles (arrows). Shown are representative sections of platelets derived from C560β3 (left panel) and R560β3 (right panel) tx mice depicting mature α-granules, vacuoles, and vesicular canals. These high magnification images showed that platelets expressing C560β3 bound, endocytosed and stored fibrinogen within α-granule in a fairly normal distribution pattern (left arrows). In striking contrast, R560β3 platelets show that fibrinogen was detected in an uneven distribution pattern bound mainly to the platelet surface (right arrows) with some endocytosis of fibrinogen present within canals and α-granules. A minimum of 100 sections were examined from each platelet sample. Isotype-matched IgG was used as a negative control for staining (not shown). Each sample was cut on a minimum of 3 separate occasions with a series of ultrathin sections subjected to immunogold labeling. bar=0.2 µm. (D) R560β3 Washed Platelets Aggregated with the Simultaneous Addition of Fibrinogen and a Mixture of Activation Agonists (ADP, epinephrine, and PAR4). C560β3 and R560β3 platelets aggregated appreciably in comparison to β3(−/−) negative control platelets, which failed to aggregate. Aggregation of β3(+/−) platelets was used as a positive control using identical conditions. The mean fluorescence intensity (MFI) of FITC-conjugated moAb to αIIb revealed that the receptor levels were similar for tx mice C560β3 (black 7±3%) and R560β3 (red and green 5±2%) compared to β3(−/−,+/−,+/+) controls assigned an arbitrary value of 0%, 50%, and 100%, respectively. Results represent platelet aggregation profiles under constant stirring from five β3(−/−,+/−) controls and eight C560β3 and six R560β3 mice. Slight variations in % aggregation profiles between R560β3 and C560 β3 samples may be due to minor differences in receptor levels. (E) Aggregation Failed when R560β3 Platelets were Incubated First with Fibrinogen for Four Minutes and Then Treated with Activation Agonists (ADP, epinephrine, and PAR4). Remarkably, with this slight change in protocol, R560β3 platelets changed shape but failed to aggregate following addition of activation agonists. This result suggests that pre-treating platelets with fibrinogen allowed immediate binding of the ligand to its receptor αIIbR560β3, thus saturating them with fibrinogen and preventing platelet-platelet aggregates from forming upon agonist simulation. As anticipated, using identical conditions, β3(−/−) platelets failed to aggregate, while C560β3 and β3(+/−) positive control platelets aggregated appreciably. The MFI at saturating concentrations of moAb to αIIb revealed that the receptor levels were similar for tx mice R560β3 (red 6±1%, green 9±1%), C560β3 (black 10±1%) when compared to β3(−/−,+/−,+/+) controls assigned an arbitrary value of (0%,50%,100%) respectively. Results represent aggregation profiles using constant stirring of platelets in a minimum of two experiments using β3(−/−,+/−,+/+) controls and one C560β3 and three R560β3 mice. Slight variations in % aggregation profiles between the two R560 samples may be due to minor differences in receptor levels, although the overall result of shape change without aggregation is consistent. (F) In vivo platelet function was examined by light microscopic analysis of fixed lung tissue stained with Trichrome following IV injection of a platelet agonist (ADP) into mice (Magnification, 400×). Platelets that aggregated into a thrombus were not detected within the pulmonary blood vessels (BV) of β3(−/−) negative control and R560β3 tx mice following infusion with 0.35 mg ADP/g body weight. In contrast, C560β3 platelets aggregated appreciably to form thrombi that were detected within the pulmonary blood vessels similar in content to the prominent thrombi that formed in the pulmonary blood vessels (BV) of β3(+/−) controls (see arrows). Trichrome stain differentially displays protein fibers (fibrotic tissue: muscle, collagen fibers, fibrin clots) and erythrocytes to allow identification of platelet aggregates within BV among the terminal bronchiole (TB), and alveolar ducts (AD). This result demonstrates that the single amino acid substitution of C560Rβ3 prevented activated platelets from undergoing normal aggregation in vivo. This result represents the outcome observed after viewing 10 sections of each lung from seven controls (four β3(−/−), three β3(+/−) mice) as well as two C560β3 and two R560β3 experimental mice 5 weeks post tx. The MFI at saturating concentrations of moAb to αIIb revealed that the receptor levels were similar for tx mice R560β3 (6±1%), C560β3 (8±2%) when compared to β3(−/−,+/,+/+) controls assigned an arbitrary value of (0%,50%,100%) respectively.

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