Collagen-induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α2β1. Adhesion and degranulation-promoting adapter protein (ADAP) regulates αIIbβ3 in platelets and αLβ2 in T cells, and is phosphorylated in GPVI-deficient platelets activated by collagen.
To determine whether ADAP plays a role in collagen-induced platelet activation and in the regulation and function of α2β1.
Using ADAP−/− mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation.
Results and Conclusions
Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP−/− platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α2β1-selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α2β1, was reduced in ADAP−/− platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP−/− platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α2β1. In addition, we found that ADAP−/− mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.
ADAP; collagen; GFOGER; GPVI; integrin α2β1; platelets
Upon contact with an appropriate surface, factor XII (FXII) undergoes autoactivation or cleavage by kallikrein. Zn2+ is known to facilitate binding of FXII and the cofactor, high molecular weight kininogen (HK), to anionic surfaces.
To investigate whether transition metals immobilized on liposome surfaces can initiate coagulation via the contact pathway.
Methods & Results
Liposomes containing a metal ion-chelating lipid (DOGS-NTA) were prepared by membrane extrusion (20% DOGS-NTA, 40% phosphatidylcholine, 10% phosphatidylserine, and 30% phosphatidylethanolamine). Ni2+ immobilized on such liposomes accelerated clotting in normal, but not FXI- or FXII-deficient plasma. Results were comparable to a commercial aPTT reagent. Charging such liposomes with other transition metals revealed differences in their procoagulant capacity, with Ni2+> Cu2+> Co2+ and Zn2+. Plasma could be depleted of FXI, FXII and HK by adsorption with Ni2+-containing beads, resulting in delayed clot times. Consistent with this, FXI, FXII and HK bound to immobilized Ni2+ or Cu2+ with high affinity as determined by surface plasmon resonance. In the presence of Ni2+-bearing liposomes, Km and kcat values derived for autoactivation of FXII and prekallikrein, as well as for activation of FXII by kallikrein or prekallikrein by FXIIa, were similar to literature values in the presence of dextran sulfate.
Immobilized Ni2+ and Cu2+ bind FXII, FXI and HK with high affinity and stimulate activation of the contact pathway, driving FXII-mediated coagulation. Activation of the contact system by immobilized transition metals may have implications during pathogenic infection or in individuals exposed to high levels of pollution.
Contact pathway; Factor XII; Factor XI; Metal ions; Coagulation; Thrombin
While heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of factor IXa inhibition to heparin activity in human plasma remains incompletely defined.
To determine the relevance of factor IXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity.
Thrombin generation was detected by fluorogenic substrate cleavage. Inhibitory potency (EC50) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), Fondaparinux, and unfractionated heparin (UFH) was determined by plotting concentration versus relative velocity index (ratio +/− heparin). Inhibition was compared under factor IX-dependent and independent conditions (0.2 or 4 pM TF, respectively) in normal plasma, and in mock- or antithrombin/factor IX-depleted plasma supplemented with recombinant factor IX.
UFH and Fondaparinux demonstrated similar potency under factor IX-dependent and independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and Fondaparinux (42-fold) demonstrated markedly increased EC50 values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (2-fold) were less affected, with an EC50 within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing factor IX/antithrombin-depleted plasma with 90 nM recombinant factor IX possessing mutations in the heparin-binding exosite. Mutated factor IX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition.
Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the factor IXa heparin-binding exosite.
In our previous in vitro study we reported a constitutively active chimeric P2Y12 receptor (cP2Y12) and found AR-C78511 is a potent inverse agonist at this receptor. The role of this cP2Y12 receptor in platelet activation and thrombosis is not clear.
To investigate the physiological implications of the constitutively active P2Y12 receptor in platelet activation, thrombus formulation and evaluate the antiplatelet activity of AR-C78511 as an inverse agonist.
Methods and Results
We generated transgenic mice conditionally and platelet-specifically expressing cP2Y12. High expression of cP2Y12 receptor in platelets increased platelet reactivity as evidenced by increased platelet aggregation in response to multiple platelet agonists. Moreover, transgenic mice displayed shortened bleeding time, more rapid and stable thrombus formation in mesenteric artery injured with FeCl3. The constitutive activity of cP2Y12 in platelets was confirmed by decreased platelet cAMP levels and constitutive Akt phosphorylation in the absence of agonists. AR-C78511 reversed the cAMP decrease in transgenic mouse platelets, and exhibited superior antiplatelet effect over AR-C69931MX in transgenic mice.
These findings further emphasize the importance of P2Y12 in platelet activation, hemostasis and thrombosis, as well as the prothrombotic role of constitutive activity of P2Y12. Our data also validates the in vivo inverse agonist activity of AR-C78511 and confirms its superior antiplatelet activity over neutral antagonist.
P2Y12; platelet; thrombosis; transgenic mice; inverse agonist
Modulation of the energy substrate metabolism may constitute a novel therapeutic intervention in the ischemic heart. AMP-activated protein kinase (AMPK) has emerged as a key regulator of favorable metabolic signaling pathways in response to myocardial ischemia. Recently, we demonstrated that activated protein C (APC) is cardioprotective against ischemia/reperfusion (I/R) injury by augmenting AMPK signaling.
The objective of this study is to determine if the APC modulation of substrate metabolism contributes to its cardioprotective effect in I/R injury.
An ex vivo working mouse heart perfusion system was used to characterize the effect of wild-type APC and its signaling-proficient mutant, APC-2Cys (has dramatically reduced anticoagulant activity), on glucose transport in the ischemic heart.
Both APC and APC-2Cys (0.2 μg/g) augment the ischemic stress-induced translocation of the glucose transporter (GLUT4) to the myocardial cell membrane, leading to increased glucose uptake and glucose oxidation in the ischemic heart (p<0.05 vs. vehicle). Both APC derivatives increased the autophagic flux in the heart following I/R. The activity of APC-2Cys in modulating these metabolic pathways was significantly higher than APC during I/R (p<0.05). Intriguingly, APC-2Cys, but not wild-type APC, attenuated the I/R-initiated fatty acid oxidation by 80% (p<0.01 vs. vehicle).
APC exerts a cardioprotective effect against I/R injury by preferentially enhancing the oxidation of glucose over fatty acids as energy substrates in the ischemic heart. Noting its significantly higher beneficial metabolic modulatory effect, APC-2Cys may be developed as a potential therapeutic drug for treating ischemic heart disease without risk of bleeding.
APC; AMPK; GLUT4; Ischemia/reperfusion; Autophagy; Cardioprotection
Tissue factor (TF) is frequently overexpressed in cancer cells and correlated with more aggressive tumor phenotypes and poor prognosis. In addition to promoting coagulation-dependent metastasis and cancer-associated thrombosis, tumor cell-expressed TF mediates direct cell signaling involving the protease activated receptor (PAR) 2. Ixolaris is a tick-derived inhibitor of the TF-FVIIa-Xa coagulation initiation complex which blocks primary tumor growth and angiogenesis in glioblastoma and melanoma models.
Here we address the anti-tumor effects of Ixolaris in TF-VIIa-PAR2 signaling-dependent breast cancer models, a xenograft model of highly aggressive human MDA-MB-231mfp cells and a syngeneic model of PAR2-deficient and replete PyMT mouse mammary carcinoma cells.
Ixolaris potently inhibited the procoagulant activity of human MDA-MB-231mfp or murine PyMT breast cancer cells. Ixolaris blocked signaling by the ternary TF-FVIIa-FXa complex, and, surprisingly, at higher concentrations also the binary TF-FVIIa complex on MDA-MB-231 cells. We show that Ixolaris interacts with certain residues in the human VIIa protease domain that are involved in PAR2 cleavage. In contrast to human VIIa, Ixolaris was a poor inhibitor of murine TF-FVIIa signaling and did not attenuate PAR2-dependent tumor growth in a syngeneic mouse model of breast cancer progression.
These data show that Ixolaris inhibits PAR2 cleavage specifically by human TF signaling complexes and suggest that Ixolaris may block tumor growth of human cell models with ectopic FVIIa expression through inhibition of direct TF-FVIIa-PAR2 signaling as well as its anticoagulant activity.
The glycoprotein (GP) Ib-IX-V complex, the von Willebrand factor receptor on platelet surface, is critically involved in hemostasis and thrombosis. GPV subunit interacts with GPIb-IX to form the GPIb-IX-V complex, but the underlying molecular basis remains unclear. It was observed earlier that efficient expression of GPV in the plasma membrane requires coexpression of GPIb-IX.
Objectives and methods
Hypothesizing that GPIb-IX stabilizes GPV through direct interaction and consequently enhances GPV surface expression, we aim in this study to identify structural elements in the complex that mediate the interaction between GPV and GPIb-IX by analyzing mutational effects on GPV surface expression in transfected Chinese hamster ovary cells.
Enhancement of GPV surface expression by GPIb-IX requires transmembrane domains of both GPV and GPIbα, as replacing GPV transmembrane domain with an unrelated poly-leucine-alanine sequence abolished the enhancing effect of GPIb-IX. Additional mutagenesis analysis of the GPV transmembrane helix identified three helical sides containing conserved polar residues as critical to efficient GPV surface expression. Similarly, replacing residues in three sides (Gly495/Ala502/Leu509, Phe491/Trp498/Val505, and Y492/L499/L506) of the GPIbα transmembrane domain to leucines preserved the surface expression level of GPIb-IX but significantly altered that of GPV.
Our results demonstrate for the first time the importance of transmembrane domains to efficient surface expression of GPV and suggest that GPV and GPIbα transmembrane domains interact with each other, contributing to assembly of the GPIb-IX-V complex.
GPIb-IX-V complex; complex assembly; transmembrane domain interaction
To determine whether an association exists between mean platelet volume (MPV) and acute myocardial infarction (AMI) and other cardiovascular events. Platelet activity is a major culprit in atherothrombotic events. MPV, which is widely available in clinical practice, is a potentially useful biomarker of platelet activity in the setting of cardiovascular disease.
Methods and Results
We performed a systematic review and meta-analysis investigating the association between MPV and AMI, all-cause mortality following myocardial infarction, and restenosis following coronary angioplasty. Results were pooled using random-effects modeling. Pooled results from 16 cross-sectional studies involving 2809 patients investigating the association of MPV and AMI indicated that MPV was significantly higher in those with AMI than those without AMI [mean difference 0.92 fL, 95% confidence interval (CI) 0.67–1.16, P < 0.001). In subgroup analyses, significant differences in MPV existed between subjects with AMI, subjects with stable coronary disease (P < 0.001), and stable controls (P < 0.001), but not vs. those with unstable angina (P = 0.24). Pooled results from three cohort studies involving 3184 patients evaluating the risk of death following AMI demonstrated that an elevated MPV increased the odds of death as compared with a normal MPV (11.5% vs. 7.1%, odds ratio 1.65, 95% CI 1.12–2.52, P = 0.012). Pooled results from five cohort studies involving 430 patients who underwent coronary angioplasty revealed that MPV was significantly higher in patients who developed restenosis than in those who did not develop restenosis (mean difference 0.98 fL, 95% CI 0.74–1.21, P < 0.001).
Elevated MPV is associated with AMI, mortality following myocardial infarction, and restenosis following coronary angioplasty. These data suggest that MPV is a potentially useful prognostic biomarker in patients with cardiovascular disease. Whether the relationship is causal, and whether MPV should influence practice or guide therapy, remains unknown.
mean platelet volume; meta-analysis; mortality; myocardial infarction; platelets
A poor biological response to clopidogrel is associated with an increased risk of major cardiovascular ischemic events (MACE). Paraoxonase 1 (PON1) enzyme activity is modulated by the PON1-Q192R variant (rs662) and was recently suggested to be strongly involved in clopidogrel bioactivation, but the influence of the PON1-Q192R variant on the risk of MACE in clopidogrel-treated patients is controversial.
To determine whether the PON1-Q192R variant influences clopidogrel biological responsiveness and the risk of MACE in patients treated with clopidogrel.
Systematic review and meta-analysis of studies of the association between the PON1-Q192R polymorphism and the biological response to clopidogrel and/or the risk of MACE during clopidogrel administration.
Seventeen studies were included. In the 12 studies of the biological response to clopidogrel (n = 5302 patients), there was no significant difference between 192QQ and 192QR + 192RR subjects, whatever the laboratory method used (global mean standardized difference = 0.10 [−0.06; 0.25], P = 0.22). Eleven studies assessed the risk of MACE, four using a case–control design (n = 2739 patients) and seven a prospective design (n = 5353 patients). Overall, MACE occurred in 19% of patients in case–control studies and in 6% of patients in prospective cohort studies, with no significant difference between 192QQ and 192QR + 192RR patients (OR = 1.28 [0.97; 1.68], P = 0.08). Similar results were obtained when study design was taken into account. Heterogeneity was mainly driven by one publication.
This meta-analysis suggests that the PON1-Q192R polymorphism has no major impact on the risk of MACE and does not alter the biological response to clopidogrel in clopidogrel-treated patients.
clopidogrel; ischemic events; paraoxonase-1; platelet function
To identify venous thromboembolism (VTE) disease-susceptibility genes.
We performed in silico genome wide association (GWAS) analyses using genotype data imputed to ~2.5 million single nucleotide polymorphisms (SNPs) from adults with objectively-diagnosed VTE (n=1503), and controls frequency-matched on age and sex (n=1459; discovery population). SNPs exceeding genome-wide significance were replicated in a separate population (VTE cases, n=1407; controls, n=1418). Genes associated with VTE were resequenced.
Seven SNPs exceeded genome-wide significance (P < 5 × 10-8); four on chromosome 1q24.2 (F5 rs6025 [Factor V Leiden], BLZF1 rs7538157, NME7 rs16861990 and SLC19A2 rs2038024) and three on chromosome 9q34.2 (ABO rs2519093 [ABO intron 1], rs495828, rs8176719 [ABO blood type O allele]). The replication study confirmed a significant association of F5, NME7, and ABO with VTE. However, F5 was the main signal on 1q24.2 as only ABO SNPs remained significantly associated with VTE after adjusting for F5 rs6025. This 1q24.2 region was shown to be inherited as a haplotype block. ABO resequencing identified 15 novel single nucleotide variations (SNV) in ABO intron 6 and the ABO 3’ UTR that were strongly associated with VTE (P < 10-4) and belonged to three distinct linkage disequilibrium (LD) blocks; none were in LD with ABO rs8176719 or rs2519093. Our sample size provided 80% power to detect odds ratios=2.0 and 1.51 for minor allele frequencies=0.05 and 0.5, respectively (α=1 × 10-8; 1% VTE prevalence).
Aside from F5 rs6025, ABO rs8176719 and rs2519093, and F2 rs1799963, additional common and high VTE-risk SNPs among whites are unlikely.
venous thromboembolism; deep vein thrombosis; pulmonary embolism; genetics; genome-wide scan; epidemiology
ADAMTS13 cleaves hyperactive ultra-large von Willebrand factor (ULVWF) multimers into smaller and less active forms. It remains unknown whether VWF-mediated inflammatory processes may play a role in the enhanced brain injury due to ADAMTS13 deficiency.
We tested the hypothesis that the deleterious effect of ADAMTS13 deficiency on ischemic brain injury is mediated through VWF-dependent enhanced vascular inflammation.
Transient focal cerebral ischemia was induced by 60 minutes of occlusion of the right middle cerebral artery. Myeloperoxidase (MPO) activity and inflammatory cytokines in the infarcted region was evaluated 23 hours after reperfusion injury. Neutrophil infiltration within the infarct and surrounding areas was quantitated by immunohistochemistry.
We report that ADAMTS13-deficient mice exhibited significantly enlarged infarct size, concordant with increased myeloperoxidase (MPO) activity, neutrophil infiltration, and expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor- α (TNF-α). In contrast, VWF-deficient mice exhibited significantly reduced MPO activity, neutrophil infiltration, and inflammatory cytokine induction, demonstrating a role for VWF in these inflammatory processes. Mice deficient for both ADAMTS13 and VWF exhibited an identical reduction of the same inflammatory parameters, demonstrating that the increased inflammation observed in ADAMTS13-deficient mice is VWF-dependent. Finally, the increased infarct size observed in ADAMTS13-deficient mice was completely abrogated by prior immunodepletion of neutrophils, demonstrating a causal role for acute inflammation in the enhanced brain injury that occurs in the setting of ADAMTS13 deficiency.
These findings provide new evidence for ADAMTS13 in reducing VWF-mediated acute cerebral inflammation following ischemic stroke.
ADAMTS13; von Willebrand factor; cerebral ischemia; inflammation
We previously found plasma levels of CD40 ligand (CD40L), chemokine (C-X-C motif) ligand 5 (CXCL5), chemokine (C-C motif) ligand 5 (CCL5), and epidermal growth factor (EGF) to be low in aplastic anemia (AA) patients and to be correlated with the platelet count.
To study the association of CD40L, CXCL5, CCL5, and EGF with platelets.
We measured cytokines in the plasma of immune thrombocytopenic purpura (ITP) and AA patients using the Luminex assay and confirmed the results in a mouse model and in vitro experiments.
Both ITP and AA showed similarly low levels of CD40L, CXCL5, CCL5, and EGF, compared with healthy controls. In ITP, levels of these proteins were significantly greater in patients with higher platelet counts than in those with lower platelet counts. In a murine thrombocytopenia model, levels of CD40L, CXCL5, CCL5, and EGF decreased with platelet count after immune-mediated destruction, while the cytokine levels increased when the platelet count recovered. In vitro, concentrations of these cytokines in the supernatants of platelet suspensions were proportional to platelet numbers, and levels in sera prepared by simple blood coagulation were equivalent to those in platelet-rich plasma-converted sera. mRNA expression for CXCL5, CCL5, and EGF was higher in platelets than in megakaryocytes, peripheral blood mononuclear cells, granulocytes, and non-megakaryocytic bone marrow cells.
Plasma CD40L, CXCL5, CCL5, and EGF are mainly platelet-derived, suggesting a role of platelets in immune responses and inflammation. Measurement of CD40L, CXCL5, CCL5, and EGF in human blood allowed testable inferences concerning physiology and pathophysiology in quantitative platelet disorders.
cytokine; immune thrombocytopenic purpura; aplastic anemia
von Willebrand Factor (VWF) is a glycoprotein that plays an important role in primary hemostasis. VWF is synthesized and stored in endothelial cells (ECs) and megakaryocytes/platelets. Plasma VWF is primarily derived from ECs and is generally believed to be essential for hemostasis. VWF synthesized in megakaryocytes is stored in platelet α-granules from which it is released following platelet activation. The relative contribution of VWF stored in ECs or megakaryocytes/platelets, or present in plasma to hemostasis is not clear.
We investigated whether EC-derived VWF plays the major role in hemostasis while the contribution of platelet-derived VWF is negligible, or if platelet-derived VWF also significantly contributes to hemostasis.
Methods and Results
Mice expressing VWF only in ECs (EC-VWF) or platelets (Plt-VWF) were created by reciprocal bone marrow transplantation between C57BL/6J (WT) and VWF knockout mice (VWF−/−). Plasma VWF levels in EC-VWF were similar to WT. Plt-VWF mice had a trace amount of VWF in their plasma while VWF levels in platelet lysate were comparable to WT. Tail bleeding time was normal in EC-VWF. Interestingly, Plt-VWF showed partially corrected bleeding time and significantly decreased blood loss volume compared to VWF−/−. Adhesion of platelets perfused over immobilized collagen under shear stress was significantly higher in both EC-VWF and Plt-VWF compared to VWF−/−.
VWF synthesized in ECs is sufficient to support hemostasis in VWF−/− mice, and VWF produced in megakaryocytes/platelets can also contribute to hemostasis in the absence of EC-derived VWF.
bone marrow transplantation; endothelial cells; platelets; von Willebrand Disease; von Willebrand Factor
Activated protein C (aPC) mediates powerful cytoprotective effects through protease activated receptor (PAR)-1 that translate into reduced harm in mouse injury models. However, it remains elusive how aPC-activated PAR1 can mediate cytoprotective effects while thrombin activation does the opposite.
We hypothesized that aPC and thrombin might induce distinct active conformations in PAR1 causing opposing effects.
We analyzed antibody binding to, and cleavage and signalling of PAR1 in either endogenously expressing endothelial or overexpressing 293T cells.
In thrombin-cleaved PAR1 neither the tethered ligand nor the hirudin like domain were available for anti-PAR1 ATAP2 and WEDE15 binding unless the tethered ligand was quenched. In contrast, aPC irreversibly prevented ATAP2 binding while not affecting WEDE15 binding. Reporter constructs with selective glutamine substitutions confirmed R41 as the only thrombin cleavage site in PAR1, whereas aPC preferentially cleaved at R46. Similarly, we report distinct cleavage sites on PAR3, K38 for thrombin and R41 for aPC. A soluble peptide corresponding to R46-cleaved PAR1 enhanced the endothelial barrier function and reduced staurosporine toxicity in endothelial as well as in 293T cells if PAR1 was expressed. Overexpression of PAR1 variants demonstrated that cleavage at R46 but not R41 is required for cytoprotective aPC signaling.
We provide a novel concept on how aPC and thrombin mediate distinct effects. We propose that the enzyme specific cleavage sites induce specific conformations which mediate divergent downstream effects. This unexpected model of PAR1 signaling might lead to novel therapeutic options for treatment of inflammatory diseases.
PAR1; thrombin; activated protein C
The development of inhibitory antibodies, referred to as inhibitors, against exogenous FVIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof-of-principle that platelet-specific expression could be successful for treating hemophilia A in the presence of inhibitory antibodies.
To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre-existing inhibitors.
Platelet-FVIII expression in pre-immunized FVIIInull mice was introduced by transplantation of lentivirus-transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined by a chromogenic assay. The transgene copy number per cell was quantitated by real time PCR. Inhibitor titer was measured by Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytic-induced venous injury model. Integration sites were analyzed by LAM-PCR.
Therapeutic levels of platelet-FVIII expression were sustained long-term without evoking an anti-FVIII memory response in the transduced pre-immunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet-FVIII expression resulting in phenotypic correction in pre-immunized secondary and tertiary recipients.
Lentivirus-mediated platelet-specific gene transfer improves hemostasis in hemophilic A mice with pre-existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitory antibodies.
Hemophilia A; Inhibitor; FVIII; Gene therapy; Platelet
CYP2C9 and VKORC1 genotypes predict therapeutic warfarin dose at initiation of therapy; however, the predictive ability of genetic information after a week or longer is unknown. Experts have hypothesized that genotype becomes irrelevant once International Normalized Ratio (INR) values are available because INR response reflects warfarin sensitivity.
We genotyped the participants in the Prevention of Recurrent Venous Thromboembolism (PREVENT) trial, who had idiopathic venous thromboemboli and began low-intensity warfarin (therapeutic INR 1.5-2.0) using a standard dosing protocol. To develop pharmacogenetic models, we quantified the effect of genotypes, clinical factors, previous doses, and INR on therapeutic warfarin dose in the 223 PREVENT participants who were randomized to warfarin and achieved stable therapeutic INRs.
A pharmacogenetic model using data from day 0 (before therapy initiation) explained 54% of the variability in therapeutic dose (R2). The R2 increased to 68% at day 7, 75% at day 14, and 77% at day 21, because of increasing contributions from prior doses and INR response. Although CYP2C9 and VKORC1 genotypes were significant independent predictors of therapeutic dose at each weekly interval, the magnitude of their predictive ability diminished over time: partial R2 of genotype was 43% at day 0, 12% at day 7, 4% at day 14, and 1% at day 21.
Over the first weeks of warfarin therapy, INR and prior dose become increasingly predictive of therapeutic dose, and genotype becomes less relevant. However, at day 7, genotype remains clinically relevant, accounting for 12% of therapeutic dose variability.
Elevated urine 11-dehydro TXB2, an indicator of persistent thromboxane generation in aspirin-treated patients, correlates with adverse cardiovascular outcome and has recently been identified as an independent risk factor for vein graft thrombosis after cardiac bypass surgery in the Reduction in Graft Occlusion Rates (RIGOR) study. The polyclonal antibody-based ELISA used to measure 11-dehydro TXB2 in these previous studies is no longer clinically available and has been supplanted by a Food and Drug Administration (FDA)-cleared second-generation monoclonal antibody-based ELISA.
To compare the laboratory and clinical performance of the first- and second-generation assays in a well-defined study population.
11-dehydro TXB2 was quantified in 451 urine samples from 229 Reduction in Graft Occlusion Rates (RIGOR) subjects using both ELISA. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and spiking studies were used to investigate discordant assay results. The association of 11-dehydro TXB2 to clinical outcome was assessed for each assay using multivariate modeling.
Median 11-dehydro TXB2 levels were higher by monoclonal antibody- compared with polyclonal antibody-based ELISA (856 vs. 399 pg mg−1 creatinine, P < 0.000001), with the latter providing values similar to UPLC-MS/MS. This discrepancy was predominantly as a result of cross-reactivity of the monoclonal antibody with 11-dehydro-2,3-dinor TXB2, a thromboxane metabolite present in a similar concentration but with a poor direct correlation with 11-dehydro TXB2. In contrast to the first-generation ELISA, 11-dehydro TXB2 measured by the monoclonal antibody-based ELISA failed to associate with the risk of vein graft occlusion.
Quantification of urine 11-dehydro TXB2 by monoclonal antibody-based ELISA was confounded by interference from 11-dehydro-2,3-dinor TXB2 which reduced the accuracy and clinical utility of this second-generation assay.
11-dehydro thromboxane B2; aspirin; ELISA; thrombosis; vein graft
Heparin-induced thrombocytopenia (HIT) is an iatrogenic disorder that occurs in a small subset of patients receiving heparin. Twenty-five per cent (or higher) of affected patients develop limb or life-threatening thrombosis. The effectiveness of therapy is incomplete and may be complicated by bleeding. HIT is caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAGs). However, antibodies with the same apparent specificity are found in many more patients without clinical disease and the reason why so few develop HIT is uncertain. We propose that HIT antibodies recognize cell surface PF4/GAG complexes on intravascular cells, including platelets and monocytes that are dynamic and mutable. Heparin removes cell surface-bound PF4 in most individuals, but removal is incomplete in those with high pre-exposure surface-bound PF4 levels. Such individuals retain critically localized cellular antigenic complexes at the time antibodies develop and are at risk to develop HIT. This article reviews the scientific basis for this model and its clinical implications.
glycosaminoglycans; heparin; immune thrombocytopenia; Platelet Factor 4; thrombocytopenia; thrombosis
Kindlin-3 is a novel integrin activator in hematopoietic cells and its deficiency leads to immune problems and severe bleeding, known as LAD-III. Our current understanding of Kindlin-3 function primarily relies on analysis of animal models or cell lines.
To understand the functions of Kindlin-3 in human primary blood cells.
Here we analyze primary and immortalized hematopoietic cells obtained from a new LAD-III patient with immune problems, bleeding, a history of anemia and abnormally shaped red blood cells.
Patient’s WBC and platelets showed defect in agonist induced integrin activation and botrocetin induced platelet agglutination. Primary leukocytes from this patient exhibited abnormal activation of beta1 integrin. Integrin activation defects were responsible for observed deficiency of botrocetin induced platelet response. Analysis of patient’s genomic DNA revealed a novel mutation in kindlin-3 gene. The mutation abolished Kindlin-3 expression in primary WBC and platelets due to abnormal splicing. Kindlin-3 is expressed in erythrocytes and its deficiency proposed to lead to abnormal shape of RBC. Immortalized patient’s WBCs expressed a truncated form of Kindlin-3 which was not sufficient to support integrin activation. Expression of Kindlin-3 cDNA in immortalized patient’s WBCs rescued integrin activation defects while overexpression of the truncated form did not.
Kindlin-3 deficiency impairs integrin function, including activation of beta 1 integrin.
Abnormalities in GPIb-IX function in kindlin-3 deficient platelets are secondary to integrin defects.
Region of Kindlin-3 encoded by Exon 11 is crucial for its ability to activate integrins in humans.
Integrins; Kindlins; Leukocyte Adhesion Deficiency; Platelets; Red Blood Cells; White Blood Cells
As a regulator of the penultimate step in the coagulation cascade, thrombin represents a principal target of direct and specific anticoagulants.
A potent thrombin inhibitor complexed with a colloidal nanoparticle was devised as a first-in-class anticoagulant with prolonged and highly localized therapeutic impact conferred by its multivalent thrombin-absorbing particle surface.
PPACK (Phe(D)-Pro-Arg-Chloromethylketone) was secured covalently to the surface of perfluorocarbon-core nanoparticle structures. PPACK and PPACK nanoparticle inhibition of thrombin were assessed in vitro via thrombin activity against a chromogenic substrate. In vivo antithrombotic activity of PPACK, heparin, non-functionalized nanoparticles, and PPACK nanoparticles was assessed through IV administration prior to acute photochemical injury of the common carotid artery. Perfluorocarbon particle retention in extracted carotid arteries from injured mice was assessed via 19F magnetic resonance spectroscopy (MRS) and imaging (MRI) at 11.7 T. APTT measurements determined the systemic effects of the PPACK nanoparticles at various times after injection.
Optical assay verified that PPACK nanoparticles exceeded PPACK’s intrinsic activity against thrombin. Application of the an in vivo acute arterial thrombosis model demonstrated that PPACK nanoparticles outperformed both heparin (p=.001) and uncomplexed PPACK (p=.0006) in inhibiting thrombosis. 19F MRS confirmed that PPACK nanoparticles specifically bound to sites of acute thrombotic injury. APTT normalized within twenty minutes of PPACK nanoparticles injection.
PPACK nanoparticles present thrombin-inhibiting surfaces at sites of acutely forming thrombi that continue to manifest local clot inhibition even as systemic effects rapidly diminish and thus represent a new platform for localized control of acute thrombosis.
imaging; inhibitors; nanotechnology; pharmacology; thrombin; thrombosis