Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential

Abstract Background Heparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin. Materials and Methods Seven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software. Results In the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT. Conclusion In these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. Figure. Figure. Disclosures Walenga: Eurofarma: Research Funding.

Download Full-text

Abstract 516: Chromosome 18p Deletion Inhibits Platelet Aggregation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.516 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Andrew D Meyer ◽

Anjana Raghunath ◽

Patricia Heard ◽

Jannine Cody

Keyword(s):

Cardiovascular Disease ◽

Platelet Aggregation ◽

Platelet Function ◽

Disease Modeling ◽

Significant Risk ◽

The United States ◽

Gene Copy ◽

Impedance Aggregometry ◽

Significant Difference ◽

Normal Controls

Inappropriate platelet function is a significant risk factor for cardiovascular disease, the leading cause of death in the United States. Although abnormal platelet function has a strong genetic component, very few human genes have been linked to platelet function. Mice with a homozygous deletion of EMILIN2 (Elastin Microfibril Interface Located Protein2) gene, located on Chromosome 18p, have a significant decrease in platelet function and clot formation. However, deletion or inactivation of only one copy of a gene is most relevant to human disease modeling. Our hypothesis is that blood samples from people with single 18p deletions that include EMILIN2 will have decreased platelet function compared to healthy individuals. We conducted a case-control study of nine adult individuals with chromosome 18p deletions matched with healthy men and women (n=20). Routine coagulation measurements were performed on a STAGO STA-R instrument. Platelet aggregation was measured with whole blood impedance aggregometry and Thromboelastography with PlateletMapping using the manufacturers’ protocols. There was no significant difference in platelet count, prothrombin time, partial thromboplastin time, d-dimer, or fibrinogen between individuals with a single 18p gene copy number and normal controls. However, platelet aggregation was impaired in individuals with 18p deletions compared to normal controls in response to collagen and arachidonic acid (ASPI), respectively ( p <0.0001, Figure 1). Moreover, Thromboelastography with PlateletMapping was decreased in individuals with 18p deletions compared to normal controls for ADP and ASPI, ( p <0.001). Individuals with one copy of 18p have decrease platelet function compared to normal controls. These results identify a novel human genetic loci linked to a specific phenotype of platelet function. Future will studies will determine if this gene can be used for diagnostic or therapeutics for cardiovascular disease.

Download Full-text

Effect of alteplase on platelet function and receptor expression

Journal of International Medical Research ◽

10.1177/0300060519829991 ◽

2019 ◽

Vol 47 (4) ◽

pp. 1731-1739 ◽

Cited By ~ 1

Author(s):

Jun Lu ◽

Peng Hu ◽

Guangyu Wei ◽

Qi Luo ◽

Jianlin Qiao ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Adenosine Diphosphate ◽

Receptor Expression ◽

Light Transmittance ◽

Dependent Manner ◽

Clot Retraction ◽

Platelet Receptors

Objective To investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function. Methods Human platelets were incubated with different concentrations of alteplase followed by analysis of platelet aggregation in response to adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid or epinephrine using light transmittance aggregometry. Platelet activation and surface levels of platelet receptors GPIbα, GPVI and αIIbβ3 were analysed using flow cytometry. The effect of alteplase on clot retraction was also examined. Results This study demonstrated that alteplase significantly inhibited platelet aggregation in response to ADP, collagen and epinephrine in a dose-dependent manner, but it did not affect ristocetin- or arachidonic acid-induced platelet aggregation. Alteplase did not affect platelet activation as demonstrated by no differences in P-selectin levels and PAC-1 binding being observed in collagen-stimulated platelets after alteplase treatment compared with vehicle. There were no changes in the surface levels of the platelet receptors GPIbα, GPVI and αIIbβ3 in alteplase-treated platelets. Alteplase treatment reduced thrombin-mediated clot retraction. Conclusions Alteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.

Download Full-text

Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4–heparin antibodies to platelets and the resultant platelet activation

Blood ◽

10.1182/blood.v96.1.182.013k18_182_187 ◽

2000 ◽

Vol 96 (1) ◽

pp. 182-187 ◽

Cited By ~ 3

Author(s):

Peter M. Newman ◽

Beng H. Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Surface Expression ◽

Dynamic Binding ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Activated Platelets ◽

Igg Binding ◽

Iodine 125 ◽

First Time

Immune heparin-induced thrombocytopenia (HIT) is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. We were able to affinity purify anti-PF4–heparin IgG (HIT IgG) from the plasma of 2 patients with HIT. Under conditions that were more physiological and sensitive than those in previous studies, we observed that this HIT IgG caused platelet aggregation on the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. We quantitated, for the first time, the binding of affinity-purified HIT iodine 125–IgG to platelets as they activated in a plasma milieu. Binding of the HIT IgG was dependent on heparin and required some degree of platelet activation. Blocking the platelet FcγRII with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. We concluded that anti-PF4–heparin IgG is the component in these HIT plasmas that induces platelet aggregation. The Fab region of HIT IgG binds to PF4–heparin on the surface of activated platelets. We propose that only then does the Fc portion of the bound IgG further activate the same or adjacent platelets through the Fc receptor. Our data support a dynamic model of platelet activation in which released PF4 enhances further antibody binding and more release.

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

Serological features of antibodies to protamine inducing thrombocytopenia and thrombosis

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2014-0664 ◽

2015 ◽

Vol 53 (2) ◽

Cited By ~ 18

Author(s):

Simon Panzer ◽

Arno Schiferer ◽

Barbara Steinlechner ◽

Ludovic Drouet ◽

Jean Amiral

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Thromboembolic Event ◽

Significant Proportion ◽

Thromboembolic Events ◽

Igg Antibodies ◽

Binding Properties ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Blood Samples

AbstractA significant proportion of patients undergoing cardiopulmonary bypass develop anti-protamine antibodies, with or without the association of thromboembolic events.We extensively investigated the serological features of protamine antibodies, which developed in six patients who were clinically suspected to have heparin-induced thrombocytopenia (HIT). Three patients had thrombotic events. Sera were tested by four different commercially available immunoassays, a heparin-platelet aggregation test, and for their binding properties to heparin, platelet factor 4 (PF4), complex heparin-PF4, protamine, and protamine complex with heparin. Sera from four patients were also tested for the capability to induce platelet activation and the formation of platelet-monocyte heterotypic aggregates.The ELISA assay Zymutest HIA was strongly positive in all cases, the HPIA Asserachrome was borderline, and the gel centrifugation test PaDGIA was positive in two tested patients. Platelet aggregation tests were negative. Using a variation of the Zymutest HIA we demonstrate that IgG antibodies bound only to protamine or protamine complex with heparin, but not to heparin or PF4 only. Sera-induced platelet P-selectin expression and the formation of platelet-monocyte aggregates. Blood samples from one patient proofed positive concomitantly with the thromboembolic event. However, serological characteristics did not differ between antibodies associated with thromboembolic events from those without.These data show that protamine-induced antibodies are specific and may induce platelet activation, which explains their association with thromboembolic events.

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

Platelet Activation Markers in Patients with Peripheral Arterial Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665429 ◽

1997 ◽

Vol 78 (06) ◽

pp. 1434-1437 ◽

Cited By ~ 33

Author(s):

Paolo Gresele ◽

Mariella Catalano ◽

Carlo Giammarresi ◽

Raul Volpato ◽

Rosanna Termini ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Arterial Disease ◽

Platelet Function Test ◽

Activation Markers ◽

Significant Difference ◽

Peripheral Arterial

SummaryPeripheral vascular disease (PVD) is an indicator of diffuse atherosclerosis and is associated with a greatly increased incidence of coronary heart and cerebrovascular disease. Although several studies have assessed whether in vivo platelet activation takes place in patients with PVD, no data are available comparing different platelet function tests in this patient population.We have compared prospectively four tests for the measurement of in vivo platelet activation (plasma βTG, plasma PF4, intraplatelet (βTG and urinary excretion of 11-dehydro-TXB2) and one in vitro platelet function test (ADP-induced platelet aggregation) in 63 well-characterized patients with intermittent claudication and in 18 age- and sex- matched healthy volunteers.No statistically significant difference was found between patients and controls for plasma βTG (20.0 ± 11.8 vs. 18.8 ± 9.0 ng/ml, respectively), plasma PF4 (5.2 ± 2.9 vs. 6.3 ± 3.5 ng/ml), βTG/PF4 ratio (4.0 ± 2.9 vs. 3.6 ± 1.8), intraplatelet pTG (4503 ± 1482 vs. 4059 ± 1065 ng/ml), and threshold aggregatory concentration of ADP (1.7 ± 0.72 vs. 1.45 ± 0.56 μM).Urinary 11-dehydro-TXB2 was instead significantly higher in the PVD group (55.4 ± 27.5 vs. 26.7 ± 7.0 ng/h, p <0.001).Our study shows that urinary 11-dehydro-TXB2 is a more sensitive index of in vivo platelet activation than the measurement of either platelet specific proteins or of in vitro platelet aggregation in patients with PVD.

Download Full-text

Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4–heparin antibodies to platelets and the resultant platelet activation

Blood ◽

10.1182/blood.v96.1.182 ◽

2000 ◽

Vol 96 (1) ◽

pp. 182-187 ◽

Cited By ~ 130

Author(s):

Peter M. Newman ◽

Beng H. Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Surface Expression ◽

Dynamic Binding ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Activated Platelets ◽

Igg Binding ◽

Iodine 125 ◽

First Time

Abstract Immune heparin-induced thrombocytopenia (HIT) is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. We were able to affinity purify anti-PF4–heparin IgG (HIT IgG) from the plasma of 2 patients with HIT. Under conditions that were more physiological and sensitive than those in previous studies, we observed that this HIT IgG caused platelet aggregation on the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. We quantitated, for the first time, the binding of affinity-purified HIT iodine 125–IgG to platelets as they activated in a plasma milieu. Binding of the HIT IgG was dependent on heparin and required some degree of platelet activation. Blocking the platelet FcγRII with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. We concluded that anti-PF4–heparin IgG is the component in these HIT plasmas that induces platelet aggregation. The Fab region of HIT IgG binds to PF4–heparin on the surface of activated platelets. We propose that only then does the Fc portion of the bound IgG further activate the same or adjacent platelets through the Fc receptor. Our data support a dynamic model of platelet activation in which released PF4 enhances further antibody binding and more release.

Download Full-text

Effect of Heparin:PMX60056 Complexes on Platelet Aggregation Induced by ADP or HIT Antibody

Blood ◽

10.1182/blood.v116.21.4386.4386 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4386-4386

Author(s):

Walter Jeske ◽

R. Eric McAllister ◽

Jeanine M. Walenga ◽

Michelle Paulus ◽

Jawed Fareed

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Anticoagulant Activity ◽

Platelet Factor ◽

Induce Platelet Aggregation ◽

Heparin Induced Thrombocytopenia ◽

Platelet Aggregometry ◽

Positively Charged

Abstract Abstract 4386 Introduction: A neutralization of anticoagulant activity occurs when heparin binds to a variety of positively charged substances such as protamine and platelet factor 4 (PF4). PMX60056 (PolyMedix, Radnor, PA) is a novel compound that is being developed as a heparin antagonist. Since heparin-PF4 complexes are antigenic, with antibodies against this complex activating platelets to trigger thrombin generation, thrombus formation and associated morbidities, it is of interest to determine whether heparin:PMX60056 complexes affect platelet function. This study compares the effect of PMX60056 and protamine, alone and complexed to heparin, on platelet function as assessed by platelet aggregometry. Materials and Method: Whole blood, collected from 10 healthy individuals, was anticoagulated with 3.2% sodium citrate and centrifuged to make platelet rich plasma (PRP). PRP was supplemented with 10 μ g/ml heparin (~1.5 IU/ml), 10 μ g/ml heparin antagonist (protamine or PMX 60056) or a complex of 10 μ g/ml heparin and 10 μ g/ml heparin antagonist. Platelet aggregation was stimulated by the addition of ADP (5 or 10 μ M final concentration) or serum from a patient with heparin-induced thrombocytopenia. Result: ADP-induced platelet aggregation was not affected by the addition of heparin, protamine, PMX 60056, or complexes of heparin with heparin antagonist. In the HIT system, heparin + HIT serum led to a significant increase in platelet aggregation vs. saline (46.7 ± 3.2 % vs. 8.2 ± 2.9%). HIT serum + heparin antagonist did not induce platelet aggregation (PMX60056: 10.2 ± 4.4%; protamine: 11.6 ± 3.5%). The aggregation responses to HIT serum + heparin (46.7 ± 3.2%), HIT serum + heparin:PMX60056 (43.6 ± 5.8%) and HIT serum + heparin:protamine (47.8 ± 3.8%) were not significantly different. Conclusion: When mixed at equigravimetric amounts, protamine and PMX60056 do not prevent formation of immune complexes consisting of HIT antibody and heparin which lead to platelet activation. Previous data from human trials has suggested that smaller amounts of PMX60056 (less than equigravimetric) may effectively neutralize heparin. Thus, it is speculated that smaller heparin:PMX60056 complexes may induce less antibody formation than larger heparin:protamine complexes. Validation of this hypothesis in animal models or clinical studies is warranted. Disclosures: McAllister: PolyMedix, Inc.: Employment.

Download Full-text

Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia

Blood Advances ◽

10.1182/bloodadvances.2016000877 ◽

2016 ◽

Vol 1 (1) ◽

pp. 62-74 ◽

Cited By ~ 24

Author(s):

Douglas B. Cines ◽

Serge V. Yarovoi ◽

Sergei V. Zaitsev ◽

Tatiana Lebedeva ◽

Lubica Rauova ◽

...

Keyword(s):

Platelet Aggregation ◽

Cell Surface ◽

Chondroitin Sulfate ◽

Platelet Activation ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Activated Platelets ◽

Key Points

Key Points Polyphosphates form antigenic complexes with PF4 that are recognized by HIT antibodies. Polyphosphate/PF4 complexes released by activated platelets can mediate platelet aggregation by HIT antibodies in the absence of heparin or cell-surface chondroitin sulfate.

Download Full-text