Interleukin-10 promoter microsatellite polymorphisms influence the immune response to heparin and the risk of heparin-induced thrombocytopenia

Introduction

Heparin-induced thrombocytopenia (HIT) results from an atypical immune response with synthesis of IgG antibodies (Abs) to platelet factor 4/heparin complexes (PF4/H), and probably involves both B and T cells. We investigated whether 3 single nucleotide polymorphisms (SNPs), rs1800896 (− 1082G/A), rs1800871 (− 819C/T) and rs1800872 (− 592C/A) and the polymorphic CA repeat microsatellites IL10R [5325CA(11_15)] and IL10G [8134CA(14_29)] are associated with the synthesis of Abs to PF4/heparin and HIT.

Materials and methods

Eighty-two patients with definite HIT and two control groups were studied. The first control group (Ab^neg) consisted of 85 patients without Abs to PF4/heparin after cardiopulmonary bypass (CPB). The second control group (Ab^pos) consisted of 84 patients who had developed significant levels of PF4-specific antibodies after CPB, but without HIT.

Results

Allele frequencies of the 3 SNPs were similar in HIT patients and controls. Fourteen alleles in IL10G (G16 to G29) and 3 alleles in IL10R (R13 to R15) were defined. The short G20 allele of IL10G was more frequent in Ab^neg patients (8.2%) than in Ab^pos (2.9%) and HIT patients (3%). It thereby appeared to protect against developing Abs to PF4/heparin (OR 0.29; 95% CI [0.12-0.70], p = 0.006). Combined haplotypes cH1/cH8 comprising the short G20 + R13 alleles were less frequent in HIT (OR 0.33; 95% CI [0.11-0.97], p = 0.036), and levels of Abs to PF4 in Ab^pos patients were lower in cH1/cH8 subjects (p = 0.019).

Conclusion

These results suggest that IL10 promoter microsatellite polymorphisms might influence the immune response against PF4/heparin and the risk of HIT.     

Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay

Background

IgG-specific anti-PF4/heparin enzyme-immunoassays (EIAs) are sensitive but not specific for platelet-activating antibodies, the cause of heparin-induced thrombocytopenia (HIT). Two features of EIA reactivity predict for presence of HIT antibodies - the magnitude of a positive result (in optical density [OD] units) and the inhibition of reactivity at high heparin concentrations - but their combined utility remains uncertain.

Objective

To determine for an IgG-specific EIA how the OD values of a positive reaction and its inhibition by high heparin can be optimally combined.

Methods

We screened 1,000 consecutive patients with suspected HIT using an IgG-specific PF4/heparin in-house EIA with and without high heparin (100 IU/mL); and by the heparin-induced platelet activation test.

Results

Platelet-activating antibodies were rarely detected (< 0.2%) when the IgG-specific EIA was negative at the conventional cut-off (OD, 0.5). However, an OD cut-off of 1.0 resulted in an unacceptable loss of sensitivity (14/83 = 17%) for detecting platelet-activating antibodies. The high heparin step increased specificity for platelet-activating antibodies from 72% to 89% without loss of sensitivity when applied to weak-positive sera (OD ≤ 1.0). However, decreased sensitivity was observed with strong-positive sera (OD > 1.0): 11/69 such sera (16%) that did not show > 40% inhibition by high heparin nevertheless contained platelet-activating antibodies.

Conclusion

Specificity of an IgG-specific EIA for detecting platelet-activating antibodies can be optimized by applying the high heparin inhibition step to weak-positive reactions (0.5- ≤ 1.0 OD). However, applying the high heparin inhibition step to strong-positive reactions (> 1.0 OD) in our in-house assay risks falsely classifying a serum as negative for platelet-activating antibodies.     

Performance characteristics of two commercially available IgG-specific immunoassays in the assessment of heparin-induced thrombocytopenia (HIT)

Background

The in vitro demonstration of antibodies against platelet factor-4/heparin (PF4/hep) complexes is an important contribution to the diagnosis of heparin-induced thrombocytopenia (HIT). The use of PF4/hep IgG-specific immunoassays enhances the specificity of HIT-investigations without any impairment of the sensitivity. Several IgG-specific immunoassays with different origin and structure of the target antigen-complex are commercially available.

Methods

Using a retrospective cohort consisting of 459 patients suspected to have HIT, we compared the performance characteristics of two commercially available IgG-specific immunoassays, GTI- (Genetic Testing Institute) and HIA-IgG-ELISA (Hyphen Biomed Research).

Results

PF4/hep antibodies were detected in 85 and 81 sera using GTI- and HIA-IgG-ELISA, respectively. OD values and clinical likelihood of patients who tested positive in one assay only were significantly lower than in those who tested positive in both immunoassays. Both IgG-specific assays showed high negative predictive values (100%) and similar but unsatisfactory positive predictive values, determined by a minimum clinical score of 5 and a positive HIPA result (41% and 43%, respectively). The implementation of a confirmatory step using excessive heparin increased the PPV of both assays, but results in a reduction of NPV in HIA-IgG-ELISA.

Conclusions

The detection of IgG antibodies alone improves the clinical usefulness of immunoassays. However, functional assays remain indispensable to avoid the overdiagnosis of HIT caused by the detection of IgG non-platelet activating antibodies. The OD value in IgG immunoassays appears to correlate with the clinical relevance of the antibodies and might be used as a predictive parameter in the assessment of HIT.     

The Feverfew plant-derived compound, parthenolide enhances platelet production and attenuates platelet activation through NF-κB inhibition

Introduction

Few treatments are available that can safely and effectively stimulate new platelet production for thrombocytopenic patients. Additionally, recipients of transfused platelets may experience an inflammatory response due to stored platelets becoming unnecessarily activated, thus creating the need for suitable agents that will dampen undesirable platelet activation. We investigated the effect of the feverfew plant-derived compound, parthenolide on platelet production and platelet activation because of its well-studied ability to induce apoptosis or differentiation in some types of cancer.

Methods

Parthenolide was used to treat human megakaryoblastic cell lines, primary human and mouse megakaryocytes. Resulting platelet production and function was measured via flow cytometry. The two most common parthenolide signaling mechanisms, oxidative stress and nuclear factor-κB inhibition, were assessed within the megakaryocytes using reactive oxygen species, glutathione and luciferase reporter assays. The influence of parthenolide on ex vivo platelet activation was tested with parthenolide pretreatment followed by collagen or thrombin activation. The resulting P-selectin surface expression and released soluble CD40 ligand was measured.

Results

Parthenolide stimulates functional platelet production from human megakaryocyte cell lines, and from primary mouse and human megakaryocytes in vitro. Parthenolide enhances platelet production via inhibition of nuclear factor-κB signaling in megakaryocytes and is independent of the parthenolide-induced oxidative stress response. Additionally, parthenolide treatment of human peripheral blood platelets attenuated activation of stimulated platelets.

Conclusion

Overall, these data reveal that parthenolide has strong potential as a candidate to enhance platelet production and to dampen undesirable platelet activation.     

Heparin modulates the conformation and signaling of platelet integrin αIIbβ3

Introduction

The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets.

Methods

We assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbβ3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the β3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbβ3, and of recombinant fragments of αIIb and β3, by surface plasmon resonance.

Results and conclusions

Heparin binding results in conformational changes in αIIbβ3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin β3 cytoplasmic domain, but the presence of heparin increased both β3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant β3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbβ3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbβ3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.     

Whole blood impedance aggregometry detects heparin-induced thrombocytopenia antibodies

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin use. IgG antibodies to complexes of platelet factor 4 (PF4) and heparin trigger the clinical manifestations of HIT. Only a subset of these antibodies will activate platelets and these can only be identified with platelet aggregation (functional) assays. Heparin-induced platelet aggregation (HIPA) and ¹⁴C-serotonin release (SRA) assays for HIT are time-consuming and complex to perform. We have developed a whole blood impedance (WBI) test using the new Multiplate^® analyser.All samples referred to our laboratory over a 10 month period were screened for heparin-PF4 antibodies by an ELISA method (Zymutest HIA IgG). The 4T's score was used to assess HIT pretest probability.Twenty antibody positive samples were further tested by all three functional assays: light transmission aggregometry (LTA), SRA and WBI. Thirteen out of twenty samples were positive by LTA (10 patients) and 15 by WBI (11 patients). SRA, considered to be the gold standard, was used as a confirmatory test and 11 were found to be positive (10 patients); four discrepant samples were weakly positive by WBI. The prevalence of a positive functional test was strongly correlated with the 4T's clinical risk score, but a small number of low-risk patients had positive functional assays.In this study, the WBI assay detected all SRA positive patients and was positive for two others suggesting greater sensitivity. The rapid and easy to perform assay may be a useful tool for haematology laboratories to detect platelet-activating HIT antibodies.     

Decreased phosphorylation of platelet vasodilator-stimulated phosphoprotein in periodontitis--a role of periodontal pathogens

Introduction

Epidemiological studies indicate an association between periodontitis and cardiovascular disease, but the underlying mechanisms are poorly understood. Vasodilator-stimulated phosphoprotein (VASP) in its phosphorylated form represents a regulator of platelet function and an indicator for the sensitivity of platelets towards physiologically relevant antagonists of platelet function.As platelets and their activation state play a central role in the development of cardiovascular disease, this study aimed to investigate the influence of periodontal disease and periodontal pathogens on intraplatelet VASP-phosphorylation and platelet function.

Material and Methods

Besides several markers of platelet activation, basal and PGE₁ induced intracellular VASP-phosphorylation were determined in platelets of periodontitis patients (n = 26) and healthy donors (n = 19). Furthermore, platelets from healthy donors were incubated with distinct periodontal pathogens and basal and PGE₁ induced VASP-phosphorylation was determined.

Results

Compared to controls, platelets of periodontitis patients showed a significant decrease in basal and PGE₁ induced VASP-phosphorylation. VASP-phosphorylation in platelets from periodontitis patients positive for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or Tannerella forsythia was significantly decreased compared to patients that were negative for these bacteria. Furthermore, VASP-phosphorylation in platelets isolated from healthy donors was affected by incubation with these periodontal pathogens.

Conclusions

Our results provide evidence that periodontitis interferes with VASP-phosphorylation in human platelets, presumably as a consequence of a direct effect of periodontitis-associated bacteria. Decreased basal and PGE₁ induced VASP-phosphorylation might represent a mechanism responsible for enhanced platelet activation in periodontitis.     

Risk factors for unfavorable clinical outcome in patients with documented heparin-induced thrombocytopenia

Background

Prognostic factors for unfavorable clinical outcome in patients with heparin-induced thrombocytopenia (HIT) are largely unknown.

Design and methods

In this multicenter, retrospective, case-control study, all HIT patients were treated with danaparoid. Study cases were HIT patients with an unfavorable clinical outcome. Controls were HIT patients who were not study cases. Unfavorable clinical outcome was defined as the occurrence of at least one of the following clinical events: death within 60 days after HIT start date, or venous or arterial thromboembolism, amputation, major bleeding, or disseminated intra-vascular coagulation between 48 hours and 60 days after HIT start date.

Results

Compared with controls (n = 65), thrombotic episodes within 48 hours of HIT start date were more frequent (59.2% versus 32.3%; p = 0.004), the median time between HIT start date and initiation of danaparoid infusion was longer (3.0 versus 1.0 days; p = 0.001), and this treatment was more frequently underdosed (43.8% versus 18.8%; p = 0.004) in study cases (n = 49). Upon multivariate analysis, all these three parameters were significant predictive factors for unfavorable clinical outcome. The adjusted odds ratios [95% confidence interval] were 6.6 [2.5-17.3] for time between HIT start date and danaparoid initiation over 48 hours, 4.3 [1.5-12.0] for danaparoid underdosing, and 3.2 [1.3-8.0] for presence of a thromboembolic episode at HIT start date.

Conclusions

This study supports the recommendations concerning the management of HIT patients, namely discontinuation of all heparin administration once the diagnosis is suspected and prompt initiation of an alternative anticoagulant drug with a strict adherence to doses specifically recommended for these patients.     

The role of thrombin receptors PAR1 and PAR4 for PAI-1 storage, synthesis and secretion by human platelets

Introduction

Arterial thrombi contain more platelets than venous thrombi and are more resistant to fibrinolysis. This resistance could partly be due to plasminogen activator inhibitor 1 (PAI-1) secreted by platelets. The aim of this study was to elucidate differences between thrombin receptors protease-activated receptor (PAR) 1 and 4 and platelet storage, secretion and synthesis of platelet PAI-1, as compared to other platelet α-granule proteins such as VEGF and endostatin.

Materials and methods

Human isolated platelets were incubated with thrombin (0.5 U/ml), PAR1-activating peptide (AP) (0.4-30 μM) or PAR4-AP (1.5-300 μM) for up to 24 hours. ELISA, western blot and fluorescence microscopy were used to measure secretion, contents and localization of PAI-1, VEGF and endostatin.

Results

Our results show that PAI-1 and VEGF might be co-localized and that endostatin does not co-localize with either PAI-1 or VEGF. PAI-1 and VEGF show a similar secretion pattern, being more sensitive to low grade PAR1 activation, but secretion was also observed with higher concentrations of PAR4-APs. PAI-1 is secreted in an active form. PAI-1 mRNA was found in platelets, and elevated levels of PAI-1 were detected after 24 hours incubation of platelets.

Conclusions

PAI-1 and VEGF, but not endostatin, might be stored in the same α-granule in human platelets. PAI-1 and VEGF also show a similar secretion pattern, being more sensitive to PAR1 than to PAR4 activation, but the secretion is not exclusively selective. Our results also show that platelet PAI-1 is increased if incubated for 24 hours, both with addition of PAR1-activating peptide and without activation, which could indicate de novo synthesis.     

Low Concentration Detergent Sclerosants Induce Platelet Activation but Inhibit Aggregation due to Suppression of GPIIb/IIIa Activation in vitro

Introduction

Sclerotherapy is associated with thromboembolic and ischemic neurological adverse events but the effects of sclerosants on platelet function are unknown. The aim of this study was to investigate the in vitro effects of detergent sclerosants Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on platelet activation and aggregation.

Materials and Methods

Whole blood and platelet rich plasma samples were incubated with sclerosants. Platelet and platelet microparticle (PMP) counts were measured by flow cytometry. Platelet activation was examined by ELISA for soluble factors (sP-selectin, von Willebrand factor, sCD40L and serotonin) and by flow cytometry for membrane-bound markers (CD62p, CD63) and cytoplasmic calcium. Platelet aggregation was assessed by PFA-100®, light transmission and impedance (Multiplate®) aggregometry, and by flow cytometry for glycoprotein (GP) Ib and GPIIb/IIIa subunits, heterodimer expression and activation (PAC-1 binding).

Results

Both agents lysed platelets at high concentrations (≥ 0.1%) but induced platelet activation at lower concentrations as evident by a rise in membrane-bound and soluble markers, cytoplasmic calcium and release of phosphatidylserine + PMP. Agonist-stimulated platelet aggregation was inhibited by both sclerosants. Membrane expression of GPIb and GPIIb/IIIa individual subunits or heterodimer was not affected by sclerosants but the activation of GPIIb/IIIa was suppressed.

Conclusion

Low concentration sclerosants activated platelets and released microparticles but inhibited platelet aggregation due to suppression of GPIIb/IIIa activation.     

The periodontal anaerobe Porphyromonas gingivalis induced platelet activation and increased aggregation in whole blood by rat model

Introduction

More and more evidence show that periodontal anaerobes contribute to pathogenesis of peripheral artery diseases. As a typical oral anaerobe that results in periodontitis, P.gingivalis aggregats platelets in PRP in vitro and participated in artery thrombosis. However, in vivo effect on platelet activation and aggregation remains unclear. This study aimed to clarify its role on platelets activation on more physiological environment, that is, on whole blood and systemic circulation.

Materials and methods

To fully estimate platelet activation, CD62P(P-selectin) expression on platelet surface and fibrinogen binding of platelet via conjugated glycoprotein GPIIb/IIIa in whole blood were assayed by flow cytometry, and platelet aggregation was measured on an impedance aggregometor. As primary study, platelet reactivity was assessed after in vitro rat whole blood incubation with P.gingivalis strain 381 in tubes, followed or not followed by ADP and arachidonic acid stimulation. In addition, PBS solution of P.gingivalis was infused into rat to produce transient bacteremia model for 5 minutes and blood samples were subjected to analysis for platelet activation in vivo.

Results

P.gingivalis could not induce rat platelet aggregation in whole blood in vitro, but increased aggregation when irritated by collagen stimulation. Flow cytometric study showed that incubation with P.gingivalis increased CD62P expression and fibrinogen binding of platelet. Moreover, further stress by 10 μmol/L ADP and 260 mmlol/L arachidonic acid yielded additional expression. As in vivo study, after P.gingivalis solution challenged, rat platelet aggregability was enhanced, and CD62P positive percentage of platelets and further reactivity to ADP stimulation improved.

Conclusion

In whole blood and in systemic circulation, P.gingivalis could induce rat platelet activation and increase aggregability transiently. The results helped to understand the mechanism underlining which P.gingivalis promoted arteriosclerosis and thrombo-embolic disorders. Further study about chronic infection with P.gingivalis on platelet activity is expected.     

New insights in heparin-induced thrombocytopenia by the use of fluid-phase assays to detect specifically platelet factor 4/heparin complex antibodies and antibody-secreting cells

Introduction

The key feature of heparin-induced thrombocytopenia (HIT) is the production of antibodies (Ab) against the platelet factor 4 (PF4)/heparin complex. These Ab are directed against neoepitopes of the PF4 tetramer, which are induced by the complex formation with heparin. To study this humoral immune response in greater detail, either in a murine immunization model or in human blood samples, reliable and specific immune assays to detect specifically Ab against the PF4/heparin complexes, but not PF4 alone are required.

Materials and Methods

We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen, PF4/heparin, is firstly captured by specific Ab, and secondly directly detected with enzyme-conjugated streptavidin.

Results

The use of this fluid-phase principle allowed a higher specificity than the traditional solid-phase enzyme-immunoassays in terms of Ab binding to murine PF4/heparin compared to murine PF4 alone. This fluid-phase approach applied to the detection of specific murine PF4/heparin Ab-secreting cells (ASC) identified the spleen as the main lymphatic organ that contributes to the PF4/heparin Ab response in mice. IgG ASC specific for PF4/heparin are very transiently detectable in mice, which might explain why anti-PF4/heparin IgG Ab typically disappear within 100 days in humans. Furthermore, this fluid-phase approach was successfully transferred to detect human PF4/heparin-specific Ab.

Conclusion

The fluid-phase principle for the specific detection of anti-PF4/heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice. At least in mice PF4/heparin antibodies are produced by transient B cells.     

A clinical-laboratory approach contributing to a rapid and reliable diagnosis of heparin-induced thrombocytopenia

Introduction

Heparin-induced thrombocytopenia (HIT) remains a very challenging diagnosis. The first objective of this study was to compare the performance of the ID-H/PF4 PaGIA^® with the Asserachrom^® HPIA ELISA. The main purpose was to evaluate the diagnostic utility of the combination of the H/PF4 PaGIA^® with the clinical “4T's” score as a screening strategy.

Materials and Methods

102 patients with clinical suspicion of HIT were classified into risk groups using the 4T's score. The presence of HIT antibodies was assessed by two immunoassays and confirmed by a functional flow cytometric assay.

Results

Comparison of the ID-H/PF4 PaGIA^® with the Asserachrom^® HPIA ELISA demonstrated a comparable technical performance, being an excellent screening test to rule out HIT (negative predictive value or NPV = 100%). According to the 4T's score, HIT was excluded in all low risk patients (NPV = 100%). ELISA optical density levels were significantly different between all risk groups (P-values < 0.01). In contrast, due to the low positive predictive value (22%) and weak positive likelihood ratio (2.6), a positive ID-H/PF4 PaGIA^® result did not considerably increase the probability of HIT.

Conclusion

Our study confirms the combination of the 4T's score with the ID-H/PF4 PaGIA^® as a reliable strategy to rule out HIT. Yet, confirming positive ID-H/PF4 PaGIA^® results by flow cytometry within 1-2 h after blood sampling remains necessary. This novel clinical-laboratory approach can contribute in a rapid and reliable way to the definite diagnosis of HIT.     

The novel P2Y12 antagonist AZD6140 rapidly and reversibly reduces platelet activation in diabetic rats

Introduction

Excessive platelet activation fundamentally contributes to cardiovascular events and mortality in patients with diabetes mellitus. Functional resistance has been described for current antiplatelet therapies in broad populations that include patients with diabetes. We investigated acute and chronic effects of AZD6140, a reversible oral rapid-onset P2Y₁₂ antagonist, on platelet reactivity in diabetic rats.

Materials and Methods

Diabetes was induced by streptozotocin injection in male Wistar rats. After 4 weeks, AZD6140 was administered (5 mg/kg by gavage) and achieved sufficient plasma levels within 30 minutes. Platelet reactivity was determined by ADP-induced P-selectin expression, aggregation and adhesion on fibrinogen coated membranes under arterial flow conditions.

Results

At 0.5 hour, AZD6140 strongly reduced ADP-induced P-selectin surface expression, inhibited ADP-induced platelet aggregation, and significantly reduced platelet adhesion to fibrinogen under arterial flow conditions. Chronic treatment with AZD6140 (10 mg/kg bid for 2 weeks, based on data obtained in the acute study) starting at day 14 reduced P-selectin surface expression on circulating platelets, indicating lower in vivo platelet activation. Platelet reactivity was improved 12 hours after the last dose, while basal platelet activity remained reduced. AZD6140 was rapidly absorbed in diabetic rats and inhibited platelet reactivity. Chronic treatment lowered in vivo platelet activation of circulating platelets.

Conclusion

AZD6140 inhibits platelet reactivity in diabetic rats rapidly and reversibly. Markers of tonic platelet activation, which were increased in diabetic rats, were lowered to levels comparable to non-diabetic rats following chronic treatment with AZD6140.     

Pennogenin tetraglycoside stimulates secretion-dependent activation of rat platelets: evidence for critical roles of adenosine diphosphate receptor signal pathways

Background

Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been demonstrated to promote hemostasis in vivo and induce platelet aggregation in vitro. Pennogenin tetraglycoside (Tg) has been identified as one of the active ingredients in TSSPs and can induce rat platelet aggregation.

Objective

To investigate the functional role of Tg in platelets and the signaling pathway mechanisms which mediate Tg-induced platelet aggregation.

Methods and results

Using scanning electron microscopy, the turbidimetric method and flow cytometry, we demonstrated that Tg induces shape change and concentration-dependently induces aggregation, dense granule secretion and a-granule secretion in rat platelets. The activation characteristics were comprehensively confirmed using transmission electron microscopy. Apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, completely inhibited Tg-induced platelet aggregation, which was not sensitive to indomethacin or SQ29548 inhibition. Furthermore, ADP receptor antagonists inhibited Tg-induced a-granule secretion, and blockade of the P2Y1 receptor prevented Tg-induced platelet shape changes. Tg-induced dense granule secretion was not affected by ADP receptor antagonists or various various pharmacological inhibitors of the intracellular effectors involved in dense granule secretion signaling pathways.

Conclusion

We identified that Tg directly induces platelet activation and demonstrated that Tg-induced platelet activation depends on dense granule secretion of ADP, which in turn activates the P2Y1 and P2Y12 receptor signaling pathways.     

Low-molecular-weight heparin inhibition in classical complement activaton pathway during pregnancy

Introduction

Low-molecular-weight heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, particularly anti-phospholipid syndrome. Nevertheless, recent studies have suggested that heparin may exert direct effects on the placental trophoblast, independently of its anticoagulant activity. In addition, heparin prevents complement activation in vivo and protects mice from pregnancy complications.

Materials and Methods

The inhibition of the classical complement activation pathway by heparin was analyzed by means of in vitro assays and in pregnant women receiving prophylaxis with therapeutic doses (40 mg/day) of subcutaneous low molecular weight heparin by haemolysis of antibody-sensitized sheep erythrocytes (CH₅₀ assay).

Results

The specific interaction between low-molecular-weight heparin and the C1q subunit of the C1 complex of the complement cascade allowed the isolation of a small subpopulation of heparin ( 8.03 ± 1.20 μg %), with an anti-activated factor X activity more than four times greater than the starting material. This subpopulation could be responsible for the in vitro inhibition of the classical complement activation pathway evaluated by the total haemolysis of antibody-sensitized sheep erythrocytes. About 60 µg/ml of low molecular weight heparin was needed to achieve 50% of haemolysis. The detection of the classical complement pathway inhibition in pregnant women treated with heparin required a first activation with aggregated human IgG.

Conclusions

We concluded that the interaction between low-molecular-weight heparin and C1q could be relevant not only in the complement-dependent, but also in the complement-independent inflammation mechanisms responsible for the prevention of pregnancy loss.     

Bivalirudin is inferior to heparin in preservation of intraoperative autologous blood

Introduction

Bivalirudin is used as an alternative to heparin in cardiac surgery, and may be superior to heparin with regard to platelet function. Bivalirudin however, is prone to cleavage by thrombin resulting in coagulation in areas of stasis.

Material and Methods

We compared the preservation of platelet function and the quality of anticoagulation in autologous blood of 26 cardiac surgical patients collected intraoperatively and anticoagulated ex vivo with either bivalirudin or heparin, with supplementation of bivalirudin over time and prevention of stasis.

Results

We found in both preservatives a reduction in ADP-induced platelet aggregation response over a period of 105 minutes (median, IQR: 73-141) as measured by Multiplate®. Supplementation of additional bivalirudin (23 ± 1.1 μg/ml/hr) and prevention of stasis was not able to prevent thrombin generation. We found a 5-fold increase in levels of prothrombin fragment 1 + 2 in bivalirudin preserved autologous blood as compared to heparin preserved blood (F₁₊₂ levels median 8.9 nM [quartile percentiles 4.2-12.4] vs 1.3 nM [0.6-2.1], P = 0.001 Mann-Whitney, n = 10).

Conclusions

Our study suggests that preservation of platelet function in autologous blood anticoagulated with bivalirudin is not a suitable alternative to heparin.     

Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers

Introduction

Flow cytometry allows the analysis of multiple antigens in a single tube at a single cell level. We present a rapid and sensitive two tube flow cytometric protocol for the detection of multiple platelet antigens and activation markers gated on a pure platelet population.

Materials and methods

The presence of platelet specific antigens was analyzed in citrated whole blood of normal platelets and from patients diagnosed with platelet abnormalities. Quiescent platelets as well as stimulated platelets were analyzed using a gating strategy based on ubiquitously expressed platelet membrane markers.A ubiquitously expressed platelet marker was combined with antibodies against the activated alpha2b-beta3 (PAC-1), Lysosomal Activated Membrane Protein (CD63) and P-selectin (CD62P).

Results

We were able to detect the platelet antigens CD36, CD41, CD42a, CD42b and CD61 in one single tube. Our approach allowed the single tube determination of PAC-1, CD63 and CD62P after activation of platelets by thrombin, collagen, ADP and PAR-1, and determination of platelet abnormalities.

Conclusions

Our two tube multi-parameter screening protocol is suited for the analysis of platelet antigens expressed on quiescent and activated platelets and allows the detection of aberrancies as found in blood of patients with thrombocytopathy such as Glanzmann Thrombasthenia, storage pool disease with diminished granule content and patients treated with clopidogrel and acetylsalicylic acid.     

Significant relationship between platelet activation and intra-media thickness of the carotid artery in patients with ischemic cerebrovascular disease

Introduction

We have studied the relationship between the ratio of activated platelets and the thickness of intima and media of the carotid artery in ischemic CVD patients in the chronic stage.

Methods

Platelet activation was assessed by means of flow cytometry of whole blood using activation-dependent monoclonal antibodies (MoAb). Forty-one MRI-proven normative subjects and 55 patients with a history of ischemic CVD were examined. The intima-media thickness of the carotid artery was measured by using B-mode ultrasound in all subjects.

Results

The appearance rates of PAC-1-positive and CD62P-positive platelets (%) were increased in ischemic CVD patients compared with those in controls (p < 0.0001, p < 0.001, respectively) The patients and controls were divided into those with atherosclerosis (Ath+), defined as intima-media thickness 1.1 mm, and those without (Ath−). There was no significant difference of PAC-1-positive platelets between the Ath− and Ath+ subgroups in either group, but there was increase in Ath− ischemic CVD patients versus Ath− control subjects (p < 0.01), and in Ath+ patients versus Ath+ controls (p < 0.05). CD62-positive platelets in the Ath+ subgroup were significantly increased versus the Ath− subgroup in both the controls (p < 0.001) and ischemic CVD patients (p < 0.05), and there was also an increase in Ath− patients versus Ath− controls (p < 0.05).

Conclusion

Platelet activation markers were increased in patients with ischemic CVD compared with controls. A significant relationship was found between increased CD62-P-positive platelets and carotid artery abnormalities in both controls and ischemic CVD patients, suggesting that platelet activation may be a potential marker for atherosclerosis.     

Deficiency of LRP8 in mice is associated with altered platelet function and prolonged time for in vivo thrombosis

Introduction

Our group has previously reported genetic studies associating polymorphisms in the low density lipoprotein receptor related protein 8 (LRP8) gene with myocardial infarction. The aim of this study was to define the role of platelet surface LRP8 in thrombosis.

Materials and Methods

Flow cytometry, aggregometry, intravital microscopy and tail bleeding assays were used to examine platelet function and hemostasis in LRP8-deficient mice and littermate controls.

Results

We demonstrated that activation of platelets from both LRP8^+/- and LRP8^-/- mice was reduced in vitro in response to either ADP or thrombin. In vivo, LRP8-hemizygous and LRP8^-/- mice demonstrated 200% and 68% increased time for carotid occlusion in response to FeCl₃ injury, respectively. Moreover, lipidated apoE3, a ligand for LRP8, inhibited platelet activation in a dose-dependent fashion. This inhibition was markedly attenuated in LRP8^-/- but not LRP8^+/- mice and did not result from membrane cholesterol efflux or a nitric oxide dependent pathway. Tail bleeding times were unaffected in both genotypes.

Conclusions

Our results suggest that LRP8 is capable of altering thrombosis without affecting normal hemostasis through mechanisms both dependent on and independent of apoE. This suggests a means whereby clot formation could be affected in humans with LRP8 gene variants.