Contaminated heparin associated with adverse clinical events and activation of the contact system

Abstract

Background: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany.

Methods: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine.

Results: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine.

Conclusions: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Figure 1. Effect of OSCS on Kallikrein Activity

Pooled human plasma samples were treated with control unfractionated heparin (UFH) or OSCS-contaminated heparin (0.025 to 250 μg per milliliter) or with chondroitin sulfate A, synthetic OSCS, or purified OSCS contaminant (0.0025 to 25 μg per milliliter). Amidolytic activity was assessed by the addition of the S-2302 chromagenic substrate (d-Pro-Phe-Arg-p-nitroaniline); the effect on kallikrein amidolytic activity is shown (Panel A). The presence of OSCS in heparin was associated with the induction of kallikrein activity. Twenty-nine samples of heparin, representing both suspect heparin lots and control lots, were analyzed in a blinded fashion for both the presence of OSCS and the ability to activate kallikrein (Panel B). The presence of OSCS was detected and quantified by one-dimensional nuclear magnetic resonance spectroscopy (see Figure 2 in the Supplementary Appendix). The percentage of each sample that was OSCS is shown below the plot. Kallikrein amidolytic activity was assessed at various concentrations of heparin; Sample 7 was not analyzed for kallikrein activity owing to the limited quantity available. ND denotes not detectable, and OD optical density. In Panel A, T bars indicate standard deviations of replicate measurements.

Figure 2. OSCS-Induced Activation of Kallikrein in Normal or Factor XII-Depleted Plasma

Control unfractionated heparin (UFH), OSCS-contaminated UFH, chondroitin sulfate A, and synthetic OSCS were incubated with normal plasma (Panel A) or with factor XII–depleted plasma (Panel B); OSCS-induced activation of kallikrein is dependent on factor XII, as indicated. Kaolin-containing buffer was evaluated as a positive control activator of the contact system. Buffer alone was included as a negative control. Amidolytic activity was assessed by the addition of the S-2302 chromagenic substrate (d-Pro-Phe-Arg-p-nitroaniline). OD denotes optical density.

Figure 3. OSCS and the Generation of Complement-Derived C5a Anaphylatoxin in Human Plasma

OSCS-contaminated unfractionated heparin (UFH) and control UFH (0.5 to 500 μg per milliliter) (Panel A) or chondroitin sulfate A, synthetic OSCS, and purified OSCS contaminant (0.005 to 50 μg per milliliter) (Panel B) were incubated with normal plasma anticoagulated with EDTA. The generation of C5a was assessed by means of ELISA. T bars indicate standard deviations of replicate measurements.

Figure 4. OSCS Induction of Complement-Derived C3a and C5a Anaphylatoxins and Its Relationship to the Contact System

Factor XII–depleted plasma was incubated with chondroitin sulfate A, OSCS, or control buffer in the presence or absence of 5 mM EDTA, zymosan (1 mg per milliliter), or aprotinin (400 IU per milliliter) (Panel A). Specific samples were reconstituted with purified factor XII, as indicated. Normal human plasma or factor XII–depleted plasma was incubated with chondroitin sulfate A, OSCS, or control buffer in the presence or absence of 5 mM EDTA, zymosan (1 mg per milliliter), or both (Panel B). C3a and C5a generation was assessed by means of ELISA. T and I bars indicate standard deviations of replicate measurements.

Figure 5. (facing page). In Vitro and In Vivo Activity of OSCS

Human, rat, rabbit, pig, and horse plasma samples were incubated with various concentrations of OSCS-contaminated unfractionated heparin (UFH) or control UFH (Panel A). Kaolin-containing buffer was tested as a positive control. Buffer alone was included as a negative control. Kallikrein amidolytic activity was assessed by the addition of the S-2302 chromogenic substrate; OSCS induces hypotension and kallikrein activity in swine (Panels B and C). Anesthetized Yorkshire crossbred pigs (three to six pigs per group) were treated with a single intravenous bolus (5 mg per kilogram) of control UFH, OSCS-contaminated UFH, chondroitin sulfate A, or synthetic OSCS. Representative data for the heart rate, the mean arterial pressure, the systolic blood pressure, and the diastolic blood pressure are shown (Panel B). EDTA-anticoagulated plasma was collected at baseline and at 5, 10, 20, 40, and 60 minutes after infusion of test samples (Panel C). OD denotes optical density. In Panels A and C, T and I bars indicate standard deviations of replicate measurements.