Anti-PF4/heparin antibodies and venous graft occlusion in postcoronary artery bypass surgery patients randomized to postoperative unfractionated heparin or fondaparinux thromboprophylaxis.

BACKGROUND: Anti-PF4/heparin antibodies are frequently generated after coronary artery bypass grafting (CABG) surgery, with platelet-activating IgG implicated in heparin-induced thrombocytopenia (HIT). It is controversial whether non-platelet-activating antibodies are associated with thrombosis. OBJECTIVES: To determine in post-CABG patients whether thromboprophylaxis using fondaparinux vs. unfractionated heparin (UFH) reduces the frequency of anti-PF4/heparin antibodies, and whether anti-PF4/heparin antibodies are associated with early graft occlusion. METHODS/PATIENTS: In a pre-planned secondary analysis of a randomized control trial (RCT) comparing fondaparinux vs. UFH thromboprophylaxis post-CABG, we determined the frequency of anti-PF4/heparin antibody formation by solid-phase enzyme-immunoassay (EIA) and of platelet-activating antibodies by serotonin-release assay (SRA); the SRA and fluid-phase EIA were used to assess fondaparinux cross-reactivity. We also examined whether anti-PF4/heparin antibodies were associated with early arterial or venous graft occlusion (6-week CT angiography). RESULTS: We found no significant difference in the frequency of antibody formation between patients who received fondaparinux vs. UFH (65.3% vs. 46.0%; P = 0.069), and no significant fondaparinux cross-reactivity. Venous graft occlusion(s) occurred in 6/26 patients who formed 'strong' IgG antibodies (≥ 1.0 optical density [OD] units and ≥ 2× baseline) vs. 3/66 who did not (P = 0.0139). In both unadjusted and adjusted analyses, strong postoperative (but not pre-operative) anti-PF4/heparin IgG responses were associated with a markedly increased risk of early venous (but not arterial) graft occlusion (adjusted OR, 9.25 [95% CI, 1.73, 49.43]; P = 0.0093); notably, none of the three SRA-positive patients developed a venous graft occlusion. CONCLUSIONS: Fondaparinux vs. UFH thromboprophylaxis postCABG does not reduce anti-PF4/heparin antibody formation. Non-platelet-activating anti-PF4/heparin IgG antibodies generated post operatively are associated with early venous graft occlusion.

Fondaparinux treatment of acute heparin-induced thrombocytopenia confirmed by the serotonin-release assay: a 30-month, 16-patient case series.

BACKGROUND: Fondaparinux is theoretically an attractive agent for the treatment of immune heparin-induced thrombocytopenia (HIT), a prothrombotic disorder caused by platelet-activating anti-platelet factor 4/heparin antibodies. Although reports of the use of fondaparinux for this indication have thus far been favorable, the diagnosis of HIT in most cases was not based on definitive laboratory confirmation of heparin-dependent, platelet-activating antibodies. OBJECTIVES: To report thrombotic and major bleeding outcomes with fondaparinux in patients with a high likelihood of having acute HIT based on clinical features and a positive result in the confirmatory platelet serotonin-release assay (SRA), a sensitive and specific test for platelet-activating HIT antibodies. METHODS/PATIENTS: We reviewed consecutive eligible patients with SRA-positive HIT (mean peak serotonin release, 91% [normal, < 20%]; mean IgG-specific PF4/heparin enzyme immunoassay result, 2.53 optical density units [normal, < 0.45 units]) in one medical center over a 30-month period who received fondaparinux for anticoagulation during acute HIT (platelet count, < 150 × 10(9) L(-1)). Where available, plasma samples were used to measure thrombin-antithrombin (TAT) complex levels. RESULTS: Sixteen patients with SRA-positive HIT received fondaparinux: 14 surgical (11 after cardiac surgery; three after vascular surgery) and two medical (acute stroke). Fifty-six per cent of patients had HIT-associated thrombosis at the time of diagnosis. No patient developed new, recurrent or progressive thrombosis; one patient developed a major bleed (calf hematoma). One patient judged to have irreversible tissue necrosis before receiving fondaparinux therapy ultimately required limb amputation. TAT complex levels were reduced within 24 h of starting fondaparinux, and 13 of 13 patients were successfully switched to warfarin. CONCLUSION: Fondaparinux shows promise for the treatment of patients with SRA-positive acute HIT.

Heparin-induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests.

BACKGROUND: Laboratory confirmation of heparin-induced thrombocytopenia (HIT) is based on detection of heparin-dependent platelet-activating antibodies. Platelet factor 4 (PF4)/heparin enzyme-immunoassays (EIA) are a widely available surrogate for platelet-activating antibodies. OBJECTIVE: Defining the optical density (OD) reactivity profiles of a PF4/heparin EIA in reference subject and patient populations and the correlation of the EIA results (expressed in OD units) with the prevalence of platelet-activating antibodies. PATIENTS/METHODS: Using quantile regression we determined the 97.5th percentile of PF4/heparin-immunoglobulin G (IgG) EIA reactivities in non-heparin-treated individuals [blood donors (n = 935)] and patients before heparin therapy (n = 1207). In patients with suspected HIT, we compared the correlation of EIA-IgG reactivities (Greifswald laboratory; n = 2821) and the heparin-induced platelet activation assay (HIPA) with the correlation of reactivities of another EIA-IgG (McMaster laboratory; n = 1956) with the serotonin-release assay (SRA). RESULTS: PF4/heparin-IgG EIA OD reactivities had a lower OD 97.5th percentile in blood donors compared with patient groups before heparin treatment (P < 0.001). The percentage of sera testing positive in the functional assays strongly correlated with PF4/heparin-IgG EIA OD reactivities in both laboratories with very similar results (correlation coefficient > 0.9) when normalized OD ranges (maximum OD divided by 10) were used instead of absolute OD values. CONCLUSIONS: Results of PF4/heparin-IgG EIA should not be reported as only positive or negative as there is no single acceptable cut-off value. Instead, reporting PF4/heparin-IgG EIA OD results in ranges allows for risk-stratified prediction for presence of platelet-activating antibodies. Use of normalized OD ranges permits a standardized approach for inter-laboratory comparisons.

Heparin-induced thrombocytopenia--therapeutic concentrations of danaparoid, unlike fondaparinux and direct thrombin inhibitors, inhibit formation of platelet factor 4-heparin complexes.

BACKGROUND: Treatment of heparin-induced thrombocytopenia (HIT), a disorder in which anti-platelet factor 4 (PF4)-heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non-heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin-mimicking pentasaccharide). PF4-heparin complexes form at optimal stoichiometric ratios. OBJECTIVES: To compare the effects of these various non-heparin anticoagulants in disrupting the formation of PF4-heparin complexes, and PF4-containing immune complexes. PATIENTS/METHODS: Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4-heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. RESULTS: Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4-heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4-transfected HEK-293 EBNA cell line expressing the PF4 receptor CXCR3-B (P = 0.0408), reduced PF4-heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4-heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4-heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. CONCLUSIONS: Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4-containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.

Quantitative interpretation of optical density measurements using PF4-dependent enzyme-immunoassays.

BACKGROUND: Many laboratories test for heparin-induced thrombocytopenia (HIT) using a PF4-dependent enzyme-immunoassay (EIA). An advantage of the EIA is its simplicity; a disadvantage is that it only indirectly detects heparin-dependent, platelet-activating antibodies ('HIT antibodies'). OBJECTIVES: To determine whether the magnitude of a positive EIA result, expressed in optical density (OD) units, predicts risk of HIT antibodies, defined as a strong-positive platelet serotonin-release assay (SRA) result (>or=50% serotonin release). PATIENTS/METHODS: We determined the risk of a strong-positive SRA result for five categories of OD reactivity (<0.40, 0.40-<1.00, 1.00-<1.40, 1.40-<2.00, and >or=2.00 OD units) using two EIAs (commercial anti-PF4/polyanion IgG/A/M and in-house anti-PF4/heparin-IgG). RESULTS: For patient sera investigated for HIT antibodies, a weak-positive result (0.40-<1.00 OD units) in either EIA indicated a low probability (or= 2.00 units. Quantifying the EIA-SRA relationship for 1553 referred patient sera, we found that for every increase of 0.50 OD units in the EIA-IgG, the risk of a strong-positive SRA result increased by OR = 6.39 [95% confidence interval (CI), 5.13, 7.95; P < 0.0001]. For every increase of 1.00 OD units in the EIA-IgG, the risk increased by OR = 40.81 (95% CI, 26.35, 63.20; P < 0.0001). CONCLUSIONS: The probability of HIT antibodies (strong-positive SRA result) inferred by a positive PF4-dependent EIA varies considerably in relation to the magnitude of the EIA result, expressed as OD values. In our laboratory, the probability of HIT antibodies being present reached >or=50% only when the OD level was >or=1.40 units.

Generation of platelet-derived microparticles and procoagulant activity by heparin-induced thrombocytopenia IgG/serum and other IgG platelet agonists: a comparison with standard platelet agonists.

Heparin-induced thrombocytopenia (HIT) is a relatively common, immunoglobulin-mediated adverse drug reaction associated with in vivo thrombin generation and both venous and arterial thrombosis. Serum and purified IgG from patients with HIT induce normal platelets to generate procoagulant platelet-derived microparticles, but the magnitude of this response in comparison with other IgG and standard platelet agonists is unknown. We describe a comparison of IgG platelet agonists, including HIT-IgG/serum, heat-aggregated IgG, and platelet-activating murine monoclonal antibodies, with standard 'strong' and 'weak' platelet agonists, and have determined their relative ability to generate platelet procoagulant activity. Using washed normal platelets as targets, we observed that HIT sera as well as other IgG agonists produced similar or even greater numbers of microparticles and procoagulant activity than the standard strong platelet agonists (thrombin, collagen, and thrombin receptor agonist peptide). The only exception was the non-physiological platelet agonist, calcium ionophore, which consistently produced a platelet procoagulant response even greater than the IgG agonists. We conclude that the IgG class of platelet agonists (including pathogenic HIT antibodies) is an effective trigger of the platelet procoagulant response comparable at least to strong physiological platelet agonists. These results help to explain the association between HIT, in vivo thrombin generation, and thrombosis.

Adherent platelet morphology on adsorbed fibrinogen: effects of protein incubation time and albumin addition.

The composition of the protein layer adsorbed to a polymer has been thought to be important for the adhesion of platelets. The state of activation of adherent platelets is an additional factor that may be a predictor of biocompatibility. Activation refers to the degree of change from discoid shape to any of several spread shapes. The conformation and orientation of adsorbed adhesive proteins, which interact with receptors on the membrane of platelets, such as fibrinogen, fibronectin, and von Willebrand factor, may also be important for platelet adhesion and activation. This work deals with the behavior of fibrinogen adsorbed to PMMA alone, where the experimental variable was incubation time with the substrate, and with adsorbed fibrinogen mixed with albumin, where the experimental variable was the molar percent of fibrinogen in the adsorption solution. Shorter protein incubation times and increased albumin levels in the initial fibrinogen adsorption solution enhanced the percentages of activated platelet morphologies and increased adsorbed fibrinogen redistribution by the platelet. Lower concentrations of albumin in the initial adsorption solution enhanced platelet adhesion numbers; fibrinogen incubation time had no effect. Together, these factors can contribute to the biocompatibility of a biomaterial through their effect on platelet adhesion and activation.

In vitro leukocyte adhesion to modified polyurethane surfaces: III. Effect of flow, fluid medium, and platelets on PMN adhesion.

The operation of filters used to remove leukocytes from red cell concentrates may depend on the adhesion and mechanical trapping of leukocytes. If adhesion is a major component of filtration then filter materials which augment leukocyte adhesion will be useful. In previous leukocyte adhesion studies, done without flow, poly(ethyleneimene) (PEI) modified polyurethane (PU) films were shown to have greater adhesion when compared with unmodified PU. Since filtration is done under flow conditions, it was decided to study PMN adhesion at a number of flow rates using an established parallel plate flow cell. The influence of divalent cations, plasma and platelets were investigated in the presence of red cells, 40% Hematocrit. The number of adherent PMNs to the PEI modified films was always substantially higher than that for the unmodified ones when the shear rate was set at 30 s-1. When using Tyrode's solution containing albumin, with or without divalent cations, a maximum in PMN adhesion was found between the shear rates of 10 and 100 s-1. With Tyrode's solution containing albumin and with 10% (v/v) plasma in saline, the addition of platelets increased PMN adhesion when divalent cations were absent. Adhesion levels with 10% (v/v) plasma in saline were reduced when compared to Tyrode's solution containing albumin without divalent cations. These results support the use of filtration conditions where the concentration of plasma is reduced and the concentration of divalent cations is increased. Detailed evaluation of filter function with flow rate is also recommended. A cell adhesion promoting polymer coating, such as PEI, may be useful in improving filter efficiency.

States in adherent platelet morphology and the processing of adsorbed protein on biomaterials.

This study evaluates the range of adherent platelet morphologies and their relationship to preadsorbed protein. Fluorescently labelled protein was used so changes in its distribution could be followed along with morphological states assessed with modulation-contrast microscopy. Our particular concern was with the quantitative relationship between the platelet and the fluorescent image. The findings from this study continue to support the idea that platelets do interact with adsorbed protein so that protein redistribution occurs and that thrombin accelerates this. Evidence is also presented to support platelet shrinkage, membrane vesicle formation and destruction as a result of thrombin. The shrinking of adherent platelets causes areas free of pre-adsorbed protein to be exposed. This process will be important determining the nature of the substrate available to cells contacting surfaces along with other adsorbed protein-related processes, e.g. reversible adsorption and post-adsorptive transitions.