Bovine Heparin Demonstrates the Same Interaction with HIT Antibodies As Porcine Heparin

Heparin, an anticoagulant widely used in numerous medical applications, is considered an essential medicine by the WHO. Due to its high volume use and that it is the parent material for low molecular weight heparins, there is potential for the raw material to be in short supply. The African swine fever epidemic in China, ongoing since August 2018, has added further restraints on heparin source material supply. At present medical grade heparin in the US is only derived from porcine intestinal mucosa; however, there are explorations into using bovine, ovine, and other sources. Bovine heparin, once common place in the US pharmaceutical sector, is again under consideration by the US FDA. This study focused on the primary immunogenic activity associated with heparin, that is heparin-induced thrombocytopenia (HIT), and how the interaction of bovine heparin with functional HIT antibodies compares to that of porcine heparin. Materials and Methods Bovine unfractionated heparin from multiple manufacturers was compared to commercial medical grade porcine heparin obtained from US pharmacies. The US Pharmacopeia porcine heparin standard was used to determine potency equivalence. Antibodies to the complex of platelet factor 4/heparin (PF4/heparin) from banked clinically confirmed HIT patient apheresis fluids were combined with heparin and donor platelet rich plasma (PRP; blood collected in citrate from volunteers after signing a consent document). Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. The platelet activation response was determined on the BioData PAP-8 Platelet Aggregometer and quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). Characterization of the biophysical interaction between varying molar ratios of human PF4 and heparin was performed using photon correlation spectroscopy (PCS) and zeta potential (Zp) measurements of PF4/heparin complexes using Zetasizer Nano ZS instrumentation and software. Differences between bovine and porcine heparin were assessed by t-test or Mann-Whitney test. Concentration-response curves were analyzed by two-way ANOVA followed by the Holm-Sidak multiple comparison test using SigmaPlot software. Results Platelet activation to PF4/heparin antibodies at bovine and porcine heparin concentrations of 0.1 U/mL (56 ± 9 % vs. 54 ± 11 % MA) and 0.4 U/mL (59 ± 10 % vs. 65 ± 8 % MA) were the same with the expected inhibition (9 ± 4% MA) at the supra-therapeutic concentration of 100 U/mL. Consistent responses were obtained across 21 lots of bovine heparin, 30 lots of porcine heparin, and 38 platelet-HIT antibody combinations. The HIT potential of bovine heparin and porcine heparin was not statistically different (p>0.05). At higher medical use doses, the platelet aggregation response in the presence of HIT antibodies was actually lower for bovine heparin than porcine heparin (0.8 U/mL, 49 ± 10 % vs. 64 ± 9 % MA, p<0.05; and 1.0 U/mL, 45 ± 11 % vs. 62 ± 9 % MA, p<0.05). By PCS, it was observed that the maximal aggregation between PF4 and either porcine or bovine heparin occurred at comparable molar ratios (7.3 ± 1.5 vs. 6.4 ± 0). Although the porcine and bovine heparins exhibited comparable molecular weights (16,333 ± 153 vs. 16,790 ± 230 Da) and polydispersities (1.19 ± 0.02 vs. 1.15 ± 0.01), porcine heparin formed somewhat larger complexes with PF4 (1113 ± 65 nm) than did bovine heparin (863 ± 68 nm). The molar ratios of PF4 to heparin at which the charge of the complex was fully neutralized (Zp = 0) was comparable for porcine and bovine heparin (9.04 ± 0.19 vs. 9.97 ± 0.65). Consistent responses were obtained across 4 lots of bovine heparin and 3 lots of porcine heparin. Conclusions Bovine heparin and porcine heparin had the same in vitro functional platelet activation response in the presence of HIT antibodies, the same potential to form complexes with human PF4, and the same associated features that make PF4 immunogenic. This investigation demonstrates that bovine heparins should have a similar immunogenic response as porcine heparin at equi-unit dosing. Current refinements in the manufacturing process for bovine and porcine heparins have led to well-characterized and purified products which is reflected in their comparable biological behavior. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Disruption of PF4/Hep Multimolecular Complex Assembly Using a Minimally Anticoagulant Heparin (ODSH)

Blood ◽

10.1182/blood.v116.21.723.723 ◽

2010 ◽

Vol 116 (21) ◽

pp. 723-723

Author(s):

Manali Joglekar ◽

Pedro Quintana ◽

Stephen Marcus ◽

Jian Liu ◽

Gowthami M. Arepally

Keyword(s):

Complex Formation ◽

Electrostatic Interactions ◽

Conflicts Of Interest ◽

Anticoagulant Activity ◽

Neutralization Assay ◽

Cross Reactivity ◽

Antibody Binding ◽

Fixed Amount ◽

Platelet Factor ◽

Molar Ratios

Abstract Abstract 723 Recent studies indicate that multimolecular complexes of platelet factor 4 (PF4) and heparin (H) are central to the pathogenesis of Heparin-Induced Thrombocytopenia (HIT). PF4/H multimolecular complexes are recognized preferentially by HIT antibodies (Rauova, Blood 2005) and are potently immunizing in a murine immunization model (Suvarna, Blood 2005). Because PF4/H multimolecular complexes assemble through non-specific electrostatic interactions, we hypothesized that disruption of PF4/H charge-dependent interactions could reduce immune mediated complications. To test this hypothesis, we employed a minimally anticoagulant compound (2-O, 3-O desulfated heparin, or ODSH, ParinGenix, Inc.) and characterized the charge-dependent interactions of murine PF4 (mPF4), ODSH and unfractionated heparin (UFH). In chromogenic assays of thrombin (IIa) generation, UFH was >80-fold more potent than ODSH in inactivating heparin (IC50 of residual IIa generation for UFH=3.1 nM v. ODSH= 259 nM, (Figure 1A). However, when equimolar amounts of UFH or ODSH (1.7 mM) were tested in a PF4 neutralization assay (Saggin, Thrombosis and Haemostasis 1992), the amount of mPF4 required to neutralize 50% of the anticoagulant activity of ODSH (IC50) was 25μg/mL, as compared to 73μg/mL for UFH (~3-fold difference), indicating that charge-dependent interactions, but not anticoagulant activity, were preserved between PF4 and ODSH (Figure 1B). When ODSH was added at 2.5, 5 or 10 fold molar excess to a fixed amount of UFH (6nM) in the PF4 neutralization assay, a proportionate increase in the amount of PF4 was needed to neutralize UFH, indicating that ODSH promotes the anticoagulant effect of UFH through preferential binding of PF4. To further characterize the biophysical interactions of PF4, ODSH and UFH, we used spectrophotometry and zeta potential to study the multimolecular complex formation (Suvarna, Blood 2007). We noted that mPF4 and ODSH formed multimolecular complexes at molar ratios of 2:1, whereas mPF4 and UFH complexes occurred at molar ratios of 1:1. When increasing concentrations of ODSH were added to pre-formed PF4/H multimolecular complexes, we noted a decrease in absorbance with increasing amounts of ODSH, indicating disruption of PF4/H multimolecular complexes (Figure 1C). However, when increasing amounts of UFH was added to preformed PF4/ODSH multimolecular complexes, a plateau in signal was noted, suggesting a higher affinity of ODSH for PF4. In PF4/H immunoassays, incubation of ODSH (1μg/mL) with HIT antibodies was effective in reducing antibody binding by >50% as compared to wells without ODSH. HIT antibodies did not recognize hPF4 (10mg/mL) in complex with ODSH (0.4-3.2 mg/mL), indicating minimal cross-reactivity of HIT antibodies with PF4/ODSH complexes (Figure 1D). In summary, we show that ODSH, a minimally anticoagulant heparin, can disrupt PF4/H multimolecular complex formation through charge dependent interactions and interfere with HIT antibody binding. These studies suggest that manipulation of PF4:H charge interactions can be a potential therapeutic strategy in the management of HIT. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Prevalence of Anti-Heparin Platelet Factor 4 Antibodies in Patients with Disseminated Intravascular Coagulation.

Blood ◽

10.1182/blood.v114.22.2094.2094 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2094-2094

Author(s):

Jawed Fareed ◽

He Zhu ◽

Josephine Cunanan ◽

Walter Jeske ◽

Debra Hoppensteadt ◽

...

Keyword(s):

Platelet Activation ◽

Disseminated Intravascular Coagulation ◽

Conflicts Of Interest ◽

Platelet Factor 4 ◽

Additional Analysis ◽

Plasma Samples ◽

Platelet Factor ◽

Intravascular Coagulation ◽

Activation Products ◽

Low Levels

Abstract Abstract 2094 Poster Board II-71 Disseminated intravascular coagulation (DIC) represents a complex syndrome with multiple pathophysiologic components. Most patients with DIC exhibit thrombocytopenic responses due to endogenous consumption of platelets. A systematic study on the prevalence on anti-heparin platelet factor 4 (AHPF4) antibodies and HIT syndrome in DIC patients has not been presented. To determine the prevalence of AHPF4 antibodies in patients with suspected DIC syndrome a total of 25 plasma samples were retrospectively analyzed utilizing the two commercially available methods (GTI, Brookfield, WI and Hyphen Biomedical, Paris, France). Out of 25 patients, 24 samples were positive for the AHPF4 antibody in the GTI method (OD>0.400), whereas only 16 were positive in the Hyphen Biomedical assay (OD>0.500). Interestingly, only 9 samples were positive in both of these assays. None of the positive samples in either the GTI or the Hyphen assay exhibited a positive 14C serotonin response. Additional analysis of these samples revealed that only 8 of these patients were previously exposed to heparin. Only 4 of the baseline samples were found to contain low levels of heparin as measured by anti-Xa method (< 0.2 U/ml). Additional analysis of these samples revealed the presence of platelet activation products such as platelet factor 4 (PF4), selectin and p-selectin. These studies suggest that circulating AHPF4 antibodies are non-functional and do not produce any thrombocytopenic responses. The elevated circulating PF4 levels and other cytokines may be contributory to the generation of these antibodies in the DIC patients. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Influence of Protein Tyrosine Phosphatase Polymorphisms on the Development of Antibodies to Platelet Factor 4 and the Risk of Heparin-Induced Thrombocytopenia.

Blood ◽

10.1182/blood.v116.21.3683.3683 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3683-3683

Author(s):

Jerôme Rollin ◽

Claire Pouplard ◽

Dorothee Leroux ◽

Marc-Antoine May ◽

Yves Gruel

Keyword(s):

Immune Response ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Critical Role ◽

Platelet Factor 4 ◽

Control Group ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Protein Tyrosine ◽

Significant Difference

Abstract Abstract 3683 Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies that activate platelets via FcgRIIa receptors. The reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H complexes, and why most patients who synthesize these antibodies do not develop HIT, have not been fully defined. The immune response in HIT involves both B and T cells, and protein tyrosine kinases (PTKs) and phosphatases (PTPs) are crucial for regulating antigen receptor-induced lymphocyte activation. Moreover, some PTPs such as CD148 and low-molecular-weight PTP (LMW-PTP) could also have a critical role in platelet activation. Dysregulation of the equilibrium between PTK and PTP function could therefore have pathologic consequences and influence the pathogenesis of HIT. Aim of the study. To investigate an association between polymorphisms affecting genes encoding 4 different PTPs i.e. CD45 (PTPRC), CD148 (PTPRJ), LYP (PTPN22) and LMW-PTP (ACP1) and the development of heparin-dependent antibodies to PF4 and HIT. Patients and methods. A cohort of 89 patients with definite HIT (positive PF4-specific ELISA and positive serotonin release assay) and two control groups were studied. The first control group (Abneg) consisted of 179 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Abpos) consisted of 160 patients who had also undergone cardiac surgery with CPB and heparin, who had all developed significant levels of PF4-specific antibodies but without HIT. Genotypes of PTPRC 77C/G (rs17612648), PTPN22 1858C/T (rs2476601), PTPRJ 2965 C/G (rs4752904) and PTPRJ 1176 A/C (rs1566734) were studied by a PCR-HRM method using the LightCycler 480 (Roche). In addition, the ACP1 A, B, C alleles were defined by combining the analysis of T/C transition at codon 43 of exon 3 (rs11553742) and T/C transition at codon 41 of exon 4 (rs11553746). Results. The frequency of PTPRC 77G and PTPN22 1858T alleles was not different in HIT patients and controls, whether they had developed antibodies to PF4 or not. The third PTP gene analyzed was ACP1, in which three alleles (A, B and C) were previously associated with the synthesis of distinct active LMW-PTP isoforms exhibiting different catalytic properties. The percentage of subjects in our study carrying the AC, BB and BC genotypes was significantly higher in the HIT and the Abpos groups than in patients without antibodies to PF4 after CPB (Abneg). In addition, the ACP1 A allele was less frequent in patients with antibodies to PF4, whether they had developed HIT (25%) or not (27.5% in Abpos controls), than in Abneg subjects (37%). The AC, BB and BC genotypes (associated in Caucasians with the highest LMW-PTP enzyme activity) therefore appeared to increase the risk of antibody formation in heparin-treated patients (OR 1.8; 95% CI 1.2–2.6, p=0.004 after comparing Abpos + HIT vs. Abneg). We also evaluated 2 SNPs affecting PTPRJ encoding CD148. No significant difference was found concerning the 2965 C/G polymorphism, but the frequency of PTPRJ 1176 AC and CC genotypes was significantly lower in the HIT (17%) than in the Abneg and Abpos groups (35%, p=0.003 and 29.5%, p=0.041, respectively). The C allele therefore appeared to provide a significant protection from the risk of HIT (OR 0.52; 95%CI 0.29–0.94, p=0.041) in patients with antibodies to PF4. Discussion-Conclusion. Recent studies have demonstrated that CD148 is a positive regulator of platelet activation by maintaining a pool of active SFKs in platelets. This non-synonym PTPRJ 1176 A/C SNP is associated with a Q276P substitution inducing a torsional stress of a fibronectin domain that is critical for the activity of CD148 and may influence the pathogenic effects of HIT Abs. This study supports the hypothesis that PTPs such as LMW-PTP and CD148 influence the immune response to heparin and the risk of HIT in patients with antibodies to PF4. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Single Center Experience with the Nanoparticle-Based Flow Immunoassay for Diagnosis of Heparin-Induced Thrombocytopenia (HIT).

Blood ◽

10.1182/blood.v120.21.2189.2189 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2189-2189

Author(s):

Susanne Macher ◽

Nazanin Sareban ◽

Camilla Drexler ◽

Gerhard Lanzer ◽

Katharina Schallmoser

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Laboratory Diagnosis ◽

Heparin Therapy ◽

Enzyme Linked Immunosorbent Assay ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Score System ◽

Single Center Experience

Abstract Abstract 2189 Heparin-induced thrombocytopenia (HIT), caused by antibodies against heparin/platelet factor 4 (HPF4) complex, is a rare but potentially serious side effect of heparin therapy where due to high mortality, rapid diagnosis is crucial. For the detection of HPF4 antibodies we compared the new nanoparticle-based lateral-flow immunoassay (LFI-HIT, Milenia Biotec, Germany) and a particle gel immunoassay (PaGIA, BioRad, Germany) with an IgG-specific-PF4/polyanion enzyme-linked immunosorbent assay (IgG-ELISA, GTI Diagnostics, USA). Sera from 121 patients (54/67 f/m, median 73 years, range 14–94) with suspected HIT were prospectively tested. The LFI-HIT and the PaGIA were evaluated visually, the IgG-ELISA was positive at an optical density (OD) cutoff > 0.4. For most of the positive samples, the functional heparin-induced platelet activation (HIPA) assay was additionally performed to detect false positive serological results and to confirm a clinically relevant HIT by in vitro platelet-activation. Regarding HIT as a clinico-pathological syndrome, characteristics for HIT were evaluated for each patient by the 4Ts scoring system and divided into high, intermediate or low risk. Results of serological analyses and OD values are summarized in the table. Ten of 121 samples were positive in the LFI-HIT, 10/10 positive in the PaGIA and 8/10 positive in the IgG-ELISA. The HIPA was tested in 9/10 samples and was positive in 8/9 samples. Of the 2 samples positive for LFI-HIT and PaGIA but negative in the ELISA, 1 was HIPA positive, 1 HIPA negative, resulting in a specificity of 88.9% for the LFI-HIT assay correlated to the HIPA. From 111/121 LFI-HIT-negative samples, 2 were positive in the PaGIA, the IgG-ELISA (OD 1.318 and 2,019) and in the HIPA. Seven of the 111 LFI-HIT negative samples were positive only in the IgG-ELISA. Due to marginal positive reactions of 5/7 samples in the ELISA with OD values between 0.4 to 0.5, only 2 LIF-HIT negative IgG-ELISA positive samples were tested by HIPA and 1/2 was positive. Based on the ELISA, the sensitivity of the LFI-HIT was 91.9% (102/111 negative samples also negative in the ELISA) in contrast to 93.1% of the PaGIA. The specificity of the LFI-HIT was 80% (LFI-HIT and IgG-ELISA positive), compared to 57.9% of the PaGIA. Notably, the clinical risk estimated by the 4Ts score system (received from 92/121 patients) did not correlate with laboratory diagnosis of HIT, probably due to inadequate evaluation. Concluding our data, a reliable exclusion of HIT by rapid testing with the LFI-HIT only seems possible with additional analysis of HPF4 antibodies by IgG-ELISA and/or HIPA assay. LFI-HIT PaGIA IgG-ELISA OD IgG-ELISA HIPA assay Median (range) Samples n=121 Pos 10 Pos 10 Pos 8 2.366 (0.902-3.000) 7/7 pos Neg 2 0.199 and 0.170 1/2 pos, 1/2 neg Neg 0 - - - - Neg 111 Pos 9 Pos 2 1.318 and 2.019 2/2 pos Neg 7 0.110 (0.054-0.139) 6/6 neg Neg 102 Pos 7 0.436 (0.404-1.463) 1/2 pos, 1/2 neg Neg 95 0.082 (0.013-0.376) Disclosures: No relevant conflicts of interest to declare.

Download Full-text

A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽

10.1182/blood.v126.23.10.10 ◽

2015 ◽

Vol 126 (23) ◽

pp. 10-10

Author(s):

Jose Perdomo ◽

Jaa Yien New ◽

Zohra Ahmadi ◽

Xing-Mai Jiang ◽

Beng H Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Immune Complexes ◽

Whole Blood ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Single Chain ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Micro Fluidics

Abstract Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions. Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec. Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1). Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition. Disclosures No relevant conflicts of interest to declare.

Download Full-text

A Proposed Role for Platelet Factor 4 in Histone Pathobiology in Sepsis

Blood ◽

10.1182/blood.v124.21.98.98 ◽

2014 ◽

Vol 124 (21) ◽

pp. 98-98

Author(s):

M. Anna Kowalska ◽

Guohua Zhao ◽

Ian Johnston ◽

Elsa Treffeisen ◽

Fatoumata Diarra ◽

...

Keyword(s):

Platelet Activation ◽

Positive Charge ◽

Conflicts Of Interest ◽

Peak Effect ◽

Platelet Factor 4 ◽

Vascular Damage ◽

Platelet Factor ◽

Competitive Binding Assay ◽

Activated Platelets ◽

Bell Shaped Curve

Abstract Sepsis is a high-risk clinical setting often resulting in multi-organ failure and death. Release of chromatin NETS (neutrophil extracellular traps) from neutrophils and the toxic role of highly-positively charged histones in late sepsis have been noted previously. Also, for NET formation to occur, peptidylarginine deiminase 4 activity must be present in the neutrophils, leading to citrullinated (cit) histones formation and loss of a portion of the positive charge. The four histones (H2A, H2B, H3 and H4) alone and as octamers of the four units tightly bind DNA. H3 and H4 histones as well as mixed octameric histones can induce a sepsis-like state in mice. One feature previously noted was that histones could inhibit activated protein C (aPC) production in the presence of thrombomodulin (TM). Since aPC generation is felt to protect against vascular damage, it was felt that this might - in part - account for the deleterious effects of histones in sepsis. We have shown that another highly-positive, small molecule, platelet factor 4 (PF4, CXCL4), which exists as a tetramer and which is stored in high concentrations in platelet alpha granules to be released in large amounts post-platelet activation, binds to the chondroitin sulfate (CS) side-chain of TM (TMCS) and enhanced aPC production along a bell-shaped curve with a peak effect around 25 µg/ml. Non-modified mixed histones had a similar bell-shaped effect on aPC generation and [histones + PF4] are additive on affecting aPC generation via TMCS. We wondered, because of this overlapping biology and the fact that significant levels of free PF4 are available in late sepsis, whether PF4 might affect other histone pathobiological pathways in late sepsis focusing on PF4’s interactions with non-modified and cit-histones. We first asked whether released PF4 might affect the binding of histones to DNA within NETS. We found that PF4 binds to DNA with greater affinity than histones in a competitive binding assay and that this effect was more marked for cit-histone consistent with its decreased positive charge. We then studied PF4 biology in three known targets of histone in sepsis. (1) In aPC generation, we examined cit-histones (either mixed, H3 or H4) relative to non-modified histones in stimulating aPC generation and found that they had a more limited effect on aPC generation with TMCS, but that again, PF4 cooperated in inducing aPC generation along a bell-shaped curve. (2) Histones are known to activate platelets (known to involve the toll-like receptor 4), likely contributing to the observed thrombocytopenia in late sepsis. We affirmed this affect with mixed histones and H4. Cit-mixed histones and cit-H4 also activated platelets in a platelet aggregation system, but much more weakly. PF4 had no effect on platelet activation by non-modified histones, but enhanced platelet activation by both cit-mixed histones and cit-H4. This was especially true for platelet activation studies with cit-H4 which on its own had nearly no affect on platelet activation though in the presence of moderate levels of PF4 (25 µg/ml), cit-H4 activated platelets as well as non-modified histones. (3) Finally, both non-modified and cit-histones activate endothelial cells (EC) by binding to their cell surfaces and likely contribute to the vascular damage of late sepsis. Using a microfluidic system involving controlled photochemical injury of the EC lining we found that PF4 enhanced the observed damage after cit-H4 exposure, but not notably after a comparable H4 exposure so that peak damage (as detected by propidium iodide staining) after cit-H4 approached that seen after H4 alone. In conclusion, NET formation involves citrullination of histones, and these modified histones likely contribute significantly to pathobiology in late sepsis. We now propose that in late sepsis, free histones, especially cit-histones, are mobilized out of NETs by PF4 because the PF4 binds DNA with higher affinity. After the histones and cit-histones are released from DNA, PF4 modifies the biology of these histones, especially the cit-histones enhancing their effects on aPC generation, platelet activation and EC injury. These studies provide additional insights of how histones achieve their pathobiological effects in sepsis. Such new insights may be critical for both understanding and monitoring clinical outcome and may lead to new therapeutic targets in sepsis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Glucose Transporter 3 in Platelets Facilitates Alpha-Granule Mediated Glucose Uptake, Driving Intragranular Glycolysis That Is Required for Platelet Degranulation and Activation

Blood ◽

10.1182/blood.v126.23.417.417 ◽

2015 ◽

Vol 126 (23) ◽

pp. 417-417

Author(s):

Fidler P.L Trevor ◽

Elizabeth Middleton ◽

Jesse W Rowley ◽

Luc Boudreau ◽

Robert A. Campbell ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Plasma Membrane ◽

Glucose Metabolism ◽

Glucose Uptake ◽

Platelet Activation ◽

Plasma Membranes ◽

Glucose Transporter ◽

Conflicts Of Interest ◽

Platelet Factor

Abstract Patients with diabetes display increased thrombosis and platelet activation. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. In vitro studies of platelets under hyperglycemic conditions suggest that glucose metabolism may lead to increased platelet activation. Platelets import glucose via two glucose transporters GLUT1, which is expressed on the plasma membrane, and GLUT3, which is expressed on the plasma membrane (15%) and the remaining 85% on α-granule membranes. Following stimulation, platelet α-granules translocate to the plasma membrane and release their cargo. To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. GLUT3 KO platelets displayed a 23% reduction in basal glucose uptake compared to littermate controls. Control platelets stimulated with thrombin displayed a significant increase in glucose uptake whereas KO platelets failed to show any change. Additionally, platelet glycogen content and glycolysis intermediates were significantly reduced in KO platelets, which exhibited reduced glycolytic rates following metabolic stress (mitochondrial uncoupling). Because GLUT3 KO platelets had only a minor decrease in glucose uptake under basal conditions, but platelet glucose metabolism was dramatically altered when stimulated, we hypothesized that under basal conditions, GLUT3 facilitates glucose uptake into α-granules, to generate glycogen and fuel intragranular glycolysis that generates the energy required for α-granule degranulation. To test this hypothesis we permeabilized platelet plasma membranes, but not α-granule membranes using saponin and incubated the platelets with C13-glucose. Under these conditions, control platelets produced 2.5-fold more C13 -lactic acid than KO platelets. In vitro, GLUT3 knockout platelets display a 90% reduction in spreading on fibrinogen and collagen matrices and significant reductions in α-granule degranulation as marked by CD62p surface translocation, platelet factor 4 release, and the persistence of α-granules observed in electron micrographs of stimulated platelets. In vivo in a KBx/N model of rheumatoid arthritis, which is dependent in part on platelet activation, GLUT3 KO mice exhibited significantly reduced severity of disease. Analysis of GLUT3 mice in models of arterial thrombosis, deep vein thrombosis and tail-bleeding indicated no alteration in thrombosis between littermate controls and KO mice. Together these data indicate that GLUT3 mediated α-granule glucose uptake is essential for platelet activation and degranulation. Moreover reducing platelet GLUT3 may ameliorate the course of rheumatoid arthritis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Aspirin-Platelet Sensitivity in Patients with Essential Thrombocythemia: Role βfibrinogen G-455-a Gene Polymorphism

Blood ◽

10.1182/blood.v126.23.5197.5197 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5197-5197

Author(s):

Rossella Rosari Cacciola ◽

Elio Cacciola Gentilini ◽

Emma Cacciola

Keyword(s):

Platelet Count ◽

Gene Polymorphism ◽

Platelet Activation ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Platelet Factor ◽

Thrombotic Risk ◽

Myeloid Neoplasm ◽

The Mean ◽

Platelet Hyperaggregation

Abstract Essential thrombocythemia (ET) is a myeloid neoplasm characterized by platelet activation and thrombotic risk. Aspirin (ASA) is the standard therapy to normal platelet hyperaggregation and to prevent the thrombosis. It is reported that thrombocythaemic patients are ASA insensitive. It is debated if inherited thrombophilia increases the thrombocythemic platelet activation and, hence, the ASA platelet insensitivity. Therefore, we evaluated βFibrinogen G-455-A gene polymorphism, as thrombophilic molecular mutation associated with increased platelet aggregation, platelet count, β-thromboglobulin (β-TG) and platelet factor 4(PF4) as markers of platelet activation, fibrinogen (Fg), platelet functional activity (PFA), as indicator of ASA platelet sensitivity, clot formation time (CFT) and the maximum clot firmness (MCF), as indicators of aspirinated platelet contribution to clot firmness. We studied 40 patients (24 men, 16 women; mean age 56 years, range 37-77) with ET according to WHO criteria. The mean duration of disease was 11 years. All patients were on ASA 100 mg once daily. The βFibrinogen G455-A genotype was determined using a commercialized polymerase chain reaction kit with sequence-specific primers. Platelets were measured by automated analyzer. β-TG and PF4 were determined by ELISA. PFA, CFT and MCF were measured by Platelet Function Analyzer (PFA-100) and by ROTEM delta, respectively. All patients had heterozygous βFibrinogen G455-A. The mean platelet count was 441±72x109/L. All patients had normal Fg (244±47 mg/dl) high β-TG and PF4 (244±15 IU/ml vs 20±11 IU/ml and 162±56 IU/ml vs 6±2 IU/ml, respectively) (p<.0001 and p<.0001, respectively), prolonged C/EPI closure time (CT, unit: s, n.v. 84-160 s) (252±48 s), normal CFT (CFT, unit: s, n.v. 30-110 s) (50±7s) and MCF (MCF, unit: mm, n.v. 50-72 mm) (71±2 mm). These findings suggest that βFibrinogen G-455-A gene polymorphism does not affect the clonal platelet hyperaggregation in ET. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Increased Thromboxane in Patients with Polycythemia Vera and Thrombosis: Evidence for Aspirin-Unsuppressible Platelet Activation In Vivo

Blood ◽

10.1182/blood-2019-122828 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5372-5372

Author(s):

Rossella Rosari Cacciola ◽

Elio Gentilini Cacciola ◽

Veronica Vecchio ◽

Emma Cacciola

Keyword(s):

Platelet Count ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Vascular Events ◽

Platelet Factor ◽

Thrombotic Risk ◽

Closure Time ◽

The Mean

Polycyhemia vera (PV) is a myeloproliferative neoplasm characterized by increased thromboxane (TX) production and thrombotic risk. It is reported that serum TXB2 concentrations in PV patients are twofold higher than healthy controls and that low-dose aspirin (ASA) therapy reduces the risk of major vascular events by 50 to 60%. To evaluate this unusual size of the effect of ASA we have studied platelet count, hematocrit (HCT), β-thromboglobulin (β-TG) and platelet factor 4 (PF4), as markers of platelet activation, TXB2, as primary indicator of platelet activation, the platelet function activity (PFA), as indicator of ASA platelet sensitivity, and the clotting time (CT), as parameter of thrombin formation. We studied 60 patients (38 men, 22 women; mean age 51 years, range 32-70) with PV according to WHO criteria. The mean duration of disease was 12 years. All patients were on ASA 100 mg once daily. All patients were on phlebotomy. None had inherited or acquired thrombotic risk factors. Of 60 patients, 30 had thrombosis (20 men, 10 women) and 30 had no thrombosis. Of 30 with thrombosis, 15 developed nonfatal myocardial infarction (10 men, 5 women) defined by chest pain of typical intensity and duration and ST-segment elevation in any limb lead on electrocardiography, 10 had nonfatal stroke (8 men, 2 women) confirmed with the use of magnetic resonance imaging, and 5 (2 men , 3 women) had deep venous thrombosis confirmed by ultrasonography. Platelet count and HCT were measured by automated analyzer. β-TG and PF4 were determined by ELISA. TXB2 was measured by radioimmunoassay technique. ASA platelet sensitivity was measured by Platelet Function Analyzer (PFA-100). CT was measured by thromboelastometry. The mean platelet count was 430±170x109/L. The mean HCT value was 42±3%. The patients with thrombosis had high β-TG, PF4 and TXB2 (110±45 IU/ml, 45±21 IU/ml, and 1.700±1.990 nmol/L, respectively), shortened C/EPI closure time (T, unit: s, n.v. 84-160 s) (55±10 s) and shortened CT (CT, unit: s. n.v. 100-240 s) (45±20 s) whereas the patients without thrombosis had normal β-TG, PF4 and TXB2 (20±11 IU/ml, 6±2 IU/ml, and 800±280 nmpl/L, respectively), prolonged C/EPI closure time (249±40 s) and normal CT (110±20 s). These findings might suggest that in PV patients and thrombotic complications might need a platelet-selective dosage of ASA. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Heparin Forms Macromolecular Complexes with Protamine and Lysozyme.

Blood ◽

10.1182/blood.v114.22.1316.1316 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1316-1316

Author(s):

Shalini Chudasama ◽

Benjamin Espinasse ◽

Mark R Wiesner ◽

Gowthami M Arepally

Keyword(s):

Complex Formation ◽

Electrostatic Interactions ◽

Conflicts Of Interest ◽

Particle Formation ◽

Biophysical Properties ◽

Platelet Factor ◽

Macromolecular Complexes ◽

Molar Ratios ◽

Colloidal Interactions ◽

Positively Charged

Abstract Abstract 1316 Poster Board I-340 The immune response in Heparin-Induced Thrombocytopenia (HIT) is directed to and initiated by large multimolecular complexes of Platelet Factor 4 (PF4) and heparin. We, and others, have previously shown that PF4 and heparin multimolecular assembly occurs through colloidal interactions, wherein heparin, a negatively charged polymeric compound, facilitates macromolecular assembly by binding and neutralizing PF4's positive charge (Suvarna, Blood 2007). In these same studies, we also demonstrated that changes in the molar ratios of the two reactants result in PF4/heparin (PF4:H) complexes with markedly altered biophysical properties and immunogenicity. Because PF4:H electrostatic interactions are non-specific, we hypothesized that other positively charged proteins would exhibit similar colloidal interactions with heparin. To test this hypothesis, we selected two positively charged proteins (protamine and lysozyme) and studied heparin-dependent complex formation by spectrophotometry (A280nm), and zeta potential (Zeta Sizer, Malvern, UK). Protamine sulfate (250, 125, 62.5, 31.2, 15.6 and 7.8 mcg/mL; Mw 5.1kDa) and lysozyme (1000, 500, 250 and 125 mcg/mL; Mw 14.3kDa) were mixed with various heparin concentrations (0-160 U/mL; activity 140U/mg; Mw 12kDa) and biophysical properties characterized by both instruments. Both protamine and lysozyme showed heparin-dependent complex formation, with peak particle formation occurring over a range of heparin concentrations (2-25 U/mL ) for both compounds. For protamine, particle formation was maximal at protamine:heparin (Pr:H) molar ratios of ∼2.5-3:1, whereas lysozyme formed peak particles at lysozyme;heparin (Ly:H) molar ratios of ∼5:1 (See figure). As with PF4:H complexes, size of complexes was dependent on mass amounts of protamine or lysozyme, with particle size increasing or decreasing in proportion to the amounts of protamine or lysozyme available for complex formation. These findings indicate that heparin is capable of forming macromolecular complexes with other proteins through charge dependent interactions. Additional in vitro and in vivo studies are underway to determine if Pr:H or Lys:H complexes exhibit cross-reactivity with PF4/heparin antibodies and if complex formation is associated with immunological consequences. Disclosures No relevant conflicts of interest to declare.

Download Full-text